Acetolactate decarboxylase

ABSTRACT

The present disclosure provides methods, compositions, apparatuses and kits comprising ALDC enzymes having a better stability and/or activity, and, optionally, the yield of ALDC enzymes which can be recovered from microorganisms is improved. In some embodiments, the present disclosure provides methods, apparatuses, compositions and kits for the use of metal ions to increase stability and/or activity, and which further can be used to recover the enzymes from microorganisms in improved yields.

CROSS-REFERENCE TO RELATED APPLICATIONS

This applications claims priority to and the benefit of United Statesprovisional patent application No. 62/165,671, filed May 22, 2015;62/166,610, filed May 26, 2015; and 62/168,406, filed May 29, 2015; eachprovisional application titled “ALDC”.

INCORPORATION BY REFERENCE OF THE SEQUENCE LISTING

The sequence listing provided in the file named“20160505_NB40736_PCT_SEQS_ST25.txt” with a size of 16,666 bytes whichwas created on May 5, 2016 and which is filed herewith, is incorporatedby reference herein in its entirety.

BACKGROUND

Diacetyl is sometimes an unwanted by-product of fermentation processesof carbohydrate containing substances, e.g. wort or grape juice.Formation of diacetyl is most disadvantageous because of its strong andunpleasant smell and in case of beer even small amounts of diacetyl ofabout 0.10 to 0.15 mg/liter has a negative effect on the flavor andtaste of the beer. During the maturation of beer, diacetyl is convertedinto acetoin by reductases in the yeast cells. Acetoin is with respectto taste and flavor acceptable in beer in much higher concentrationsthan diacetyl.

Acetolactate decarboxylase (ALDC) can also be used as an enzyme toprevent the formation of diacetyl. α-acetolactate can be converted intoacetoin by adding an ALDC enzyme during fermentation. However, ALDC canbe unstable at fermenting conditions, especially those of fermentingworts with low malt content.

The purpose of the present invention is to provide ALDC enzymes having abetter stability and/or activity, and, optionally, the yield of ALDCenzymes which can be recovered from microorganisms is improved.

SUMMARY OF THE INVENTION

The present disclosure provides compositions and processes for ALDCenzymes.

Aspects and embodiments of the compositions and methods are set forth inthe following separately numbered paragraphs.

1. A composition comprising an acetolactate decarboxylase (ALDC) enzymeand zinc, where the zinc is present at a concentration of about 1 μM toabout 200 mM.

2. The composition of paragraph 1, where the zinc is present at aconcentration of about 10 μM to about 150 mM, or about 20 μM to about120 mM, or about 25 μM to about 100 mM, or about 25 μM to about 50 mM,or about 25 μM to about 20 mM, or about 25 μM to about 50 μM, or about100 μM to about 20 mM, or about 250 μM to about 20 mM, or about 500 μMto about 20 mM, or about 1 mM to about 20 mM, or about 1 mM to about 10mM, or about 1 mM to about 5 mM.

3. The composition of any preceding paragraph, where the zinc is presentat a concentration of about 100 μM to about 10 mM.

4. The composition of any preceding paragraph, where the zinc is presentat a concentration of about 1 mM to about 5 mM. The composition of anypreceding paragraph, wherein the molar ratio of zinc to ALDC enzyme ishigher than 1; or 2:1 or higher; or 10:1 or higher; or 20:1 or higher;or 30:1 or higher; or 60:1 or higher.

5. The composition of any preceding paragraph, where the ALDC enzyme isan ALDC derivative.

6. The composition of paragraph 5, where the ALDC derivative is an ALDCenzyme treated with glutaraldehyde.

7. The composition of paragraph 6, where the ALDC enzyme is treated withglutaraldehyde at a concentration corresponding to about 0.1 to about 5g of glutaraldehyde per g of pure ALDC enzyme.

8. The composition of any preceding paragraph, where the activity of theALDC enzyme is in the range of 950 to 2500 Units per mg of protein.

9. The composition of any preceding paragraph, where the activity of theALDC enzyme is in the range of 1000 to 2500 Units per mg of protein.

10. The composition of any preceding paragraph further comprising atleast one additional enzyme or enzyme derivative selected from the groupconsisting of acetolactate reductoisomerases, acetolactate isomerases,amylase, glucoamylase, hemicellulase, cellulase, glucanase, pullulanase,isoamylase, endo-glucanase and related beta-glucan hydrolytic accessoryenzymes, xylanase, xylanase accessory enzymes (for example,arabinofuranosidase, ferulic acid esterase, and xylan acetyl esterase)and protease.

11. The composition of any preceding paragraph, where the ALDC enzyme isfrom Lactobacillus casei, Brevibacterium acetylicum, Lactococcus lactis,Leuconostoc lactis, Enterobacter aerogenes, Bacillus subtilis, Bacillusbrevis, Lactococcus lactis DX, or Bacillus licheniformis.

12. The composition of any preceding paragraph, where the ALDC enzyme isfrom Bacillus brevis or Bacillus licheniformis.

13. The composition of any preceding paragraph, where the ALDC enzymehas an amino acid sequence having at least 80% identity with any oneselected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 andSEQ ID NO: 8 or any functional fragment thereof.

14. Use of the composition according to any preceding paragraph infermentation (such as beer and/or wine and/or cider and/or perry and/orsake fermentation).

15. A method for increasing the activity and/or stability of an ALDCenzyme comprising adding zinc at a concentration of about 1 μM to about200 mM. The method of paragraph 15 for increasing the activity and/orstability of an ALDC enzyme in a composition comprising ALDC whereinsaid method comprises the step of adding zinc to the composition so thatsaid zinc is present in said composition at a concentration of about 1μM to about 200 mM.

16. The method of paragraph 15, where the zinc is present in saidcomposition at a concentration of about 1 μM to about 300 μM, or about 6μM to about 300 μM, or about 1 μM to about 50 μM; or about 1 μM to about25 μM, or about 10 μM to about 150 mM, or about 20 μM to about 120 mM,or about 25 μM to about 100 mM, or about 25 μM to about 50 mM, or about25 μM to about 20 mM, or about 25 μM to about 50 μM, or about 100 μM toabout 20 mM, or about 250 μM to about 20 mM, or about 500 μM to about 20mM, or about 1 mM to about 20 mM, or about 1 mM to about 10 mM, or about1 mM to about 5 mM.

17. The method of paragraphs 15 or 16, where the zinc is present in saidcomposition at a concentration of about 100 μM to about 10 mM.

18. The method of paragraphs 15 or 16, where the zinc is present in saidcomposition at a concentration of about 1 mM to about 5 mM. The methodof paragraphs 15 or 16, where the molar ratio of zinc to ALDC enzyme ishigher than 1; or 2:1 or higher; or 10:1 or higher; or 20:1 or higher;or 30:1 or higher; or 60:1 or higher in said composition.

19. The method of paragraph 15 where the zinc is added to a cultivationmedia as a supplement during the production of the ALDC enzyme by anALDC producing host cell. A method for increasing the activity and/orstability of an ALDC enzyme in a cultivation media comprising an ALDCproducing host cell wherein said method comprises the step of addingzinc to the cultivation media as a supplement during the production ofthe ALDC enzyme by the ALDC producing host cell.

20. The method of paragraph 19, where the zinc is added at aconcentration of 1 μM to about 1 mM.

21. The method of paragraph 20, where the zinc is added at aconcentration of 25 μM to about 150 μM, or about 60 μM to about 150 μM.

22. The method of any one of paragraphs 19-21, where the host cell is aBacillus host cell.

23. The method of paragraph 22, where the Bacillus host cell is Bacillussubtilis. 24. The method of paragraph 15, where the zinc is added in afermentation media and/or maturation media during fermentation of abeverage (such as beer and/or wine and/or cider and/or perry and/or sakefermentation). A method for increasing the activity and/or stability ofan ALDC enzyme in a fermentation media and/or maturation mediacomprising an ALDC producing host cell wherein said method comprises thestep adding zinc to the fermentation media and/or maturation mediaduring fermentation of a beverage (such as beer and/or wine and/or ciderand/or perry and/or sake fermentation).

25. The method of paragraph 24, where the zinc is added at aconcentration of about 1 μM to about 300 μM, or about 6 μM to about 300μM, or about 1 μM to about 50 μM; or about 1 μM to about 25 μM.

26. The method of any one of paragraphs 24, 25 and the currentparagraph, where the zinc is added as a composition comprising ALDC andzinc, wherein the zinc is present in the composition at a concentrationof 1 mM to about 5 mM. The method of any one of paragraphs 24, 25 andthe current paragraph, where the zinc is added as a compositioncomprising ALDC and zinc, where the molar ratio of zinc to ALDC enzymein the composition is higher than 1; or 2:1 or higher; or 10:1 orhigher; or 20:1 or higher; or 30:1 or higher; or 60:1 or higher. 27. Acultivation media for an ALDC producing host cell comprising zinc at aconcentration of about 1 μM to about 1 mM; preferably said cultivationmedia comprises an ALDC producing host cell.

28. The cultivation media of paragraph 27, comprising zinc atconcentration of about 25 μM to about 150 μM.

29. The cultivation media of paragraph 27, where the zinc is added at aconcentration of 60 μM to about 150 μM.

30. A beer and/or wine and/or cider and/or perry and/or sakefermentation media and/or maturation media comprising an ALDC enzyme andzinc at a concentration of about 0.1 μM to about 200 mM, A beer and/orwine and/or cider and/or perry and/or sake fermentation media and/ormaturation media comprising a composition comprising an ALDC enzyme andzinc wherein said composition comprises zinc at a concentration of about0.1 μM to about 200 mM, preferably 1 μM to about 200 mM.

31. The beer and/or wine and/or cider and/or perry and/or sakefermentation media and/or maturation of paragraph 30, wherein saidcomposition comprises zinc at a concentration of about 0.1 μM to about300 μM, preferably 1 μM to about 300 μM. The beer and/or wine and/orcider and/or perry and/or sake fermentation media and/or maturation ofparagraph 30, comprising wherein said composition comprises zinc at aconcentration of about 6 μM to about 300 μM, or about 1 μM to about 50μM, or about 6 μM to about 50 μM, or about 6 μM to about 25 μM. The beerand/or wine and/or cider and/or perry and/or sake fermentation mediaand/or maturation of paragraph 30 and the current paragraph, where thezinc and the ALDC enzyme are added in a composition, wherein the zinc ispresent in the composition at a concentration of about 1 mM to about 20mM, such as 1 mM to about 5 mM. The beer and/or wine and/or cider and/orperry and/or sake fermentation media and/or maturation of paragraph 30or the current paragraph, where the zinc and the ALDC enzyme are addedin a composition, where the molar ratio of zinc to ALDC enzyme in thecomposition is higher than 1; or 2:1 or higher; or 10:1 or higher; or20:1 or higher; or 30:1 or higher; or 60:1 or higher.

32. The beer and/or wine and/or cider and/or perry and/or sakefermentation media and/or maturation of paragraph 30 or 31, where theactivity of the ALDC enzyme is in the range of 1000 to 2500 Units per mgof protein.

33. The beer and/or wine and/or cider and/or perry and/or sakefermentation media and/or maturation of any one of paragraphs 30-32,further comprising at least one additional enzyme or enzyme derivativeselected from the group consisting of acetolactate reductoisomerases,acetolactate isomerases, amylase, glucoamylase, hemicellulase,cellulase, glucanase, pullulanase, isoamylase, endo-glucanase andrelated beta-glucan hydrolytic accessory enzymes, xylanase, xylanaseaccessory enzymes (for example, arabinofuranosidase, ferulic acidesterase, and xylan acetyl esterase) and protease.

34. A composition comprising an ALDC enzyme, where the ALDC enzyme is inthe range of 1000 to 2500 Units per mg of protein.

35. The composition of paragraph 34, where the ALDC enzyme is fromLactobacillus casei, Brevibacterium acetylicum, Lactococcus lactis,Leuconostoc lactis, Enterobacter aerogenes, Bacillus subtilis, Bacillusbrevis, Lactococcus lactis DX, or Bacillus licheniformis.

36. The composition of paragraph 34, where the ALDC enzyme is fromBacillus brevis or Bacillus licheniformis.

37. The composition of any one of paragraphs 34-36, where the ALDCenzyme has an amino acid sequence having at least 80% identity with anyone selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7and SEQ ID NO: 8 or any functional fragment thereof.

38. The composition of any one of paragraphs 34-37, where zinc ispresent at a concentration of about 1 μM to about 200 mM, preferablyabout 100 μM to about 200 mM. The composition of any one of paragraphs34-37, where the molar ratio of zinc to ALDC enzyme in the compositionis higher than 1; or 2:1 or higher; or 10:1 or higher; or 20:1 orhigher; or 30:1 or higher; or 60:1 or higher.

39. A method for beer and/or wine and/or cider and/or perry and/or sakeproduction comprising adding an ALDC enzyme and zinc during the beerand/or wine and/or cider and/or perry and/or sake production, where thezinc is present at a concentration of about 0.1 μM to about 300 μM,preferably 1 μM to about 300 μM. The method of paragraph 39 for beerand/or wine and/or cider and/or perry and/or sake production comprisingadding an ALDC enzyme and adding zinc to a media (such as a fermentationand/or a maturation media) for the beer and/or wine and/or cider and/orperry and/or sake during said beer and/or wine and/or cider and/or perryand/or sake production so that said zinc is present in said media at aconcentration of about 0.1 μM to about 300 μM, such as about 6 μM toabout 300 μM.

40. The method of paragraph 39, where the zinc is present in said mediaat a concentration of about 0.1 μM to about 50 μM. The method ofparagraph 39, where the zinc is present in said media at a concentrationof about 6 μM to about 300 μM, or 1 μM to about 50 μM, or about 6 μM toabout 50 μM, or about 6 μM to about 25 μM.

41. The method of paragraph 39, where the zinc and the ALDC enzyme areadded in a composition, wherein zinc is present in said composition at aconcentration of about 1 mM to about 5 mM. The method of paragraph 39,where the zinc and the ALDC enzyme are added in a composition, where themolar ratio of zinc to ALDC enzyme in the composition is higher than 1;or 2:1 or higher; or 10:1 or higher; or 20:1 or higher; or 30:1 orhigher; or 60:1 or higher. A method for beer and/or wine and/or ciderand/or perry and/or sake production comprising adding a compositioncomprising an ALDC enzyme and zinc to a media (such as a fermentationand/or a maturation media) for the beer and/or wine and/or cider and/orperry and/or sake during said beer and/or wine and/or cider and/or perryand/or sake production wherein (i) zinc is present in the composition ata concentration of about 1 μM to about 200 mM, preferably about 1 mM toabout 5 mM; or (ii) the molar ratio of zinc to ALDC enzyme in thecomposition is higher than 1; or 2:1 or higher; or 10:1 or higher; or20:1 or higher; or 30:1 or higher; or 60:1 or higher.

42. The method of any one of paragraphs 39-41, where the ALDC enzyme andthe zinc are added during a fermentation process or a maturationprocess.

43. The method of any one of paragraphs 39-42, where the ALDC enzyme isadded at a concentration of about 0.5 g to about 10 g per hectoliter ofbeer and/or wine and/or cider and/or perry and/or sake ferment.

44. The method of any one of paragraphs 39-43, where the ALDC enzyme isadded at a concentration of about 1 g to about 5 g per hectoliter ofbeer and/or wine and/or cider and/or perry and/or sake ferment.

45. The method of any one of paragraphs 39-44, where the activity of theALDC enzyme is in the range of 1000 to 2500 Units per mg of protein.

46. The method of any one of paragraphs 39-45, where the ALDC enzyme isfrom Lactobacillus casei, Brevibacterium acetylicum, Lactococcus lactis,Leuconostoc lactis, Enterobacter aerogenes, Bacillus subtilis, Bacillusbrevis, Lactococcus lactis DX, or Bacillus licheniformis.

47. The method of any one of paragraphs 39-45, where the ALDC enzyme isfrom Bacillus brevis or Bacillus licheniformis.

48. The method of any one of paragraphs 39-45, where the ALDC enzyme hasan amino acid sequence having at least 80% identity with any oneselected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 andSEQ ID NO: 8 or any functional fragment thereof.

49. A method for increasing the activity and/or stability of an ALDCenzyme comprising adding a metal ion at a concentration of about 1 μM toabout 200 mM, preferably about 100 μM to about 200 mM. The method ofparagraph 49 for increasing the activity and/or stability of an ALDCenzyme in a composition comprising ALDC wherein said method comprisesthe step of adding a metal ion to the composition at a concentration ofabout 1 μM to about 200 mM, preferably about 100 μM to about 200 mM.

50. The method of paragraph 49, where the atomic radius for the metalion is about 140 pm to about 165 pm.

51. The method of paragraph 50, where the atomic radius for the metalion is about 140 pm to about 150 pm.

52. The method of paragraph 51, where the atomic radius for the metalion is about 142 pm to about 146 pm.

53. The method of paragraph 49, where the metal ion is selected from thegroup consisting of Zn²⁺, Mg²⁺, Mn²⁺, Co²⁺, Cu²⁺, Ca²⁺, Ba²⁺ and Fe²⁺and combinations thereof.

54. The method of paragraph 53, where the metal ion is selected from thegroup consisting of Zn²⁺, Mn²⁺, and Co²⁺.

55. The method of paragraph 54, where the metal ion is Zn²⁺.

56. The method of any one of paragraphs 49-55, where the ALDC enzyme isfrom Lactobacillus casei, Brevibacterium acetylicum, Lactococcus lactis,Leuconostoc lactis, Enterobacter aerogenes, Bacillus subtilis, Bacillusbrevis, Lactococcus lactis DX, or Bacillus licheniformis.

57. The method of any one of paragraphs 49-55, where the ALDC enzyme isfrom Bacillus brevis or Bacillus licheniformis.

58. The method of any one of paragraphs 49-55, where the ALDC enzyme hasan amino acid sequence having at least 80% identity with any oneselected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 andSEQ ID NO: 8 or any functional fragment thereof.

59. A composition comprising an acetolactate decarboxylase (ALDC) enzymeand a metal ion, where the metal ion is present at a concentration ofabout 1 μM to about 200 mM, preferably about 100 μM to about 200 mM.

60. The composition of paragraph 59, where the atomic radius for themetal ion is about 140 pm to about 165 pm.

61. The composition of paragraphs 59, where the atomic radius for themetal ion is about 140 pm to about 150 pm.

62. The composition of paragraph 61, where the atomic radius for themetal ion is about 142 pm to about 146 pm.

63. The composition of paragraph 59, where the metal ion is selectedfrom the group consisting of Zn²⁺, Mg²⁺, Mn²⁺, Co²⁺, Cu²⁺, Ca²⁺, Ba²⁺and Fe²⁺ and combinations thereof.

64. The composition of paragraph 63, where the metal ion is selectedfrom the group consisting of Zn²⁺, Mn²⁺, and Co²⁺.

65. The composition of paragraph 64, where the metal ion is Zn²⁺.

66. A method for decomposing acetolactate comprising the step oftreating a substrate with an ALDC enzyme and a metal ion, where themetal ion is present at a concentration of about 1 μM to about 200 mM.The method of paragraph 66 for decomposing acetolactate comprising thestep of treating a substrate with a composition comprising an ALDCenzyme and a metal ion so that the metal ion is present in saidcomposition at a concentration of about 1 μM to about 200 mM. Preferablysaid substrate is a carbohydrate containing substrate such as a wort ora fruit juice. Preferably said substrate is a fermentation and/ormaturation media.

67. The method of paragraph 66, where the metal ion is present in saidcomposition at a concentration of about 10 μM to about 150 mM, or about20 μM to about 120 mM, or about 25 μM to about 100 mM, or about 25 μM toabout 50 mM, or about 25 μM to about 20 mM, or about 100 μM to about 20mM, or about 250 μM to about 20 mM, or about 1 mM to about 20 mM, orabout 1 mM to about 5 mM.

68. The method of paragraph 66, where the metal ion is present in saidsubstrate (such as a fermentation and/or maturation media) at aconcentration of about 1 μM to about 500 μM, or about 1 μM to about 300μM, or about 6 μM to about 300 μM, or about 1 μM to about 100 μM, orabout 1 μM to about 50 μM, or about 1 μM to about 25 μM, or about 6 μMto about 50 μM, or about 6 μM to about 25 μM, or about 25 μM to about 50μM.

69. The method of paragraph 66, where the metal ion and the ALDC areadded in a composition, wherein said metal ion is present in saidcomposition at a concentration of about 1 mM to about 5 mM. The methodof paragraph 66, where the metal ion and the ALDC are added in acomposition, where the molar ratio of the metal ion to ALDC enzyme inthe composition is higher than 1; or 2:1 or higher; or 10:1 or higher;or 20:1 or higher; or 30:1 or higher; or 60:1 or higher.

70. The method of any one of paragraphs 66-69, where the metal ion isselected from the group consisting of Zn²⁺, Mg²⁺, Mn²⁺, Co²⁺, Cu²⁺,Ca²⁺, Ba²⁺ and Fe²⁺ and combinations thereof.

71. The method of any one of paragraphs 66-70, where the metal ion isselected from the group consisting of Zn²⁺, Mn²⁺, and Co²⁺.

72. The method of any one of paragraphs 66-71, where the metal ion isZn²⁺.

73. The method of any one of paragraphs 66-72, where the substrate istreated during a beer and/or wine and/or cider and/or perry and/or sakefermentation or maturation process.

74. The method of any one of paragraphs 66-72, where the ALDC enzyme isfrom Lactobacillus casei, Brevibacterium acetylicum, Lactococcus lactis,Leuconostoc lactis, Enterobacter aerogenes, Bacillus subtilis, Bacillusbrevis, Lactococcus lactis DX, or Bacillus licheniformis.

75. The method of any one of paragraphs 66-72, where the ALDC enzyme isfrom Bacillus brevis or Bacillus licheniformis.

76. The method of any one of paragraphs 66-72, where the ALDC enzyme hasan amino acid sequence having at least 80% identity with any oneselected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 andSEQ ID NO: 8 or any functional fragment thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

A better understanding of the features and advantages of the presentinvention will be obtained by reference to the following detaileddescription that sets forth illustrative embodiments, in which theprinciples of the invention are utilized, and the accompanying drawingsof which:

FIG. 1 shows a plasmid map of alrA(CB)RIHI-aldB for expression ofAcetolactate Decarboxylase, aldB

FIG. 2 shows a graph depicting the activity of ALDC relative to 0 mMzinc vs concentration of zinc in mM.

FIG. 3 shows SDS-PAGE with aldB samples containing varying concentrationof ZnSO₄. Gel A—Lane 1) Molecular weight marker; Lane 2-7) BSA standard;Lane 8-9) AldB with 0 mM ZnSO₄; Lane 10-11) AldB with 0.25 mM ZnSO₄;Lane 12-13) AldB with 0.5 mM ZnSO₄; Lane 14-15) AldB with 1.0 mM ZnSO₄;Lane 16-17) AldB with 2 mM ZnSO₄; Lane 18-19) AldB with 5 mM ZnSO₄; Lane20-21) AldB with 7.5 mM ZnSO₄; Lane 22-23) AldB with 10 mM ZnSO₄ andLane 24-25) AldB with 20 mM ZnSO₄. Gel B—Lane 1) Molecular weightmarker; Lane 2-7) BSA standard; Lane 8-11) AldB with 0 mM ZnSO₄; Lane12-15) AldB with 20 mM ZnSO₄; Lane 16-17) AldB with 40 mM ZnSO₄; Lane18-19) AldB with 60 mM ZnSO₄; Lane 20-21) AldB with 80 mM ZnSO₄; Lane22-23) AldB with 100 mM ZnSO₄ and Lane 24-25) AldB with 120 mM ZnSO₄.

FIG. 4 shows SDS-PAGE with purification of aldB produced in B. subtilis.Lane 1) crude aldB ferment; 2-3) purified aldB from Source15Q. Molecularweight marker is shown to the left and in-between lane 1 and 2.

FIG. 5 shows development of vicinal diketones (VDK) during malt-basedfermentations in the presence or absence of 0.03 U/mL wort aldB enzymevariants with different specific activity: A) 919 U/mg, B) 1103 U/mg andC) 1552 U/mg aldB. The VDK development (sum of diacetyl and 2,3pentanedione) was followed during the 7 days of fermentation at 14° C.The average VDK values are calculated from duplicate samples and labelsare shown as insert in figure.

FIG. 6 shows VDK development in presence aldB enzyme (0.04 U/mL wort)during malt-based fermentations with different levels of Zn²⁺ in thewort (see labels). The VDK development (sum of diacetyl and2,3-pentanedione) was followed during the 7 days of fermentation at 14°C. The average VDK values are calculated from duplicate samples andlabels are shown as insert in figure.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure provides methods, compositions, apparatuses andkits comprising ALDC enzymes having a better stability and/or activity,and, optionally, the yield of ALDC enzymes which can be recovered frommicroorganisms is improved. In some embodiments, the present disclosureprovides methods, apparatuses, compositions and kits for the use ofmetal ions to increase stability and/or activity, and, optionally, whichfurther can be used to recover the enzymes (e.g. ALDC enzymes) frommicroorganisms in improved yields.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Singleton, et al., DICTIONARYOF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20 ED., John Wiley and Sons, NewYork (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OFBIOLOGY, Harper Perennial, N.Y. (1991) provide one of skill with ageneral dictionary of many of the terms used in this disclosure.

This disclosure is not limited by the exemplary methods and materialsdisclosed herein, and any methods and materials similar or equivalent tothose described herein can be used in the practice or testing ofembodiments of this disclosure. Numeric ranges are inclusive of thenumbers defining the range. Unless otherwise indicated, any nucleic acidsequences are written left to right in 5′ to 3′ orientation; amino acidsequences are written left to right in amino to carboxy orientation,respectively.

The headings provided herein are not limitations of the various aspectsor embodiments of this disclosure which can be had by reference to thespecification as a whole. Accordingly, the terms defined immediatelybelow are more fully defined by reference to the specification as awhole.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “aprotease” includes a plurality of such enzymes and reference to “thefeed” includes reference to one or more feeds and equivalents thereofknown to those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that such publicationsconstitute prior art to the claims appended hereto.

All patents and publications referred to herein are incorporated byreference.

ALDC

In some aspects the invention provides ALDC enzymes having a betterstability and/or activity, and, optionally, the yield of ALDC enzymeswhich can be recovered from microorganisms is improved. The terms“better stability” and “increased stability” as used herein refer to anALDC enzyme whose ALDC activity is maintained for a longer period oftime when in the presence of a metal ion (such as Zn²⁺) when compared tothe ALDC activity of the enzyme in the absence of the metal ion. Theterms “better activity” and “increased activity” as used herein refer toan ALDC enzyme having an increased ALDC activity when in the presence ofa metal ion (such as Zn²⁺) when compared to the ALDC activity of theenzyme in the absence of the metal ion. The term “improved” inconnection to the yield of ALDC enzyme refers to an increase in the ALDCactivity which is produced when a host microorganism is in the presenceof a metal ion (such as Zn²⁺) compared to the ALDC activity producedwhen the host microorganism is in the absence of the metal ion. Withoutwishing to be bound by theory, the metal ion (such as Zn²⁺) can be addedduring and/or after the culture process (e.g. ALDC production) in orderto increase stability and/or increase activity and/or to increase yieldof ALDC enzymes. The terms “host cell”, “host microorganism”, “strain”and “microorganism” may be used interchangeably herein.

Acetolactate decarboxylase (ALDC) is an enzyme that belongs to thefamily of carboxy lyases, which are responsible for cleavingcarbon-carbon bonds. Acetolactate decarboxylase catalyzes the conversionof 2-acetolactate (also known as 2-hydroxy-2-methyl-3-oxobutanoate) to2-acetoin and releases CO₂. The terms “ALDC” and “ALDC enzyme” may beused interchangeably herein.

Acetolactate decarboxylase enzymes catalyze the enzymatic reactionbelonging to the classification EC 4.1.1.5 (acetolactate decarboxylaseactivity) and gene ontology (GO) term ID of GO: 0047605. The GO term IDspecifies that any protein characterized as having this associated GOterm encodes an enzyme with catalytic acetolactate decarboxylaseactivity.

Various acetolactate decarboxylase genes (such as alsD or aldB), whichencode acetolactate decarboxylase enzymes, are known in the art. ThealsD gene, which encodes ALDC enzyme, may be derived or derivable fromBacillus subtilis. The aldB gene, which encodes ALDC enzyme, may bederived or derivable from Bacillus brevis. The alsD gene, which encodesALDC enzyme, may be derived or derivable from Bacillus licheniformis.UNIPROT accession number Q65E52.1 is an example of an ALDC enzyme.UNIPROT accession number Q65E52.1 is an example of an ALDC enzymederived or derivable from Bacillus licheniformis. Examples ofacetolactate decarboxylase genes include but are not limited togi|375143627|ref|YP_005006068.11 acetolactate decarboxylase [Niastellakoreensis OR20-10]; gi|361057673|gb|AEV96664.1|acetolactatedecarboxylase [Niastella koreensis OR20-10];gi|218763415|gb|ACL05881.1|acetolactate decarboxylase [Desulfatibacillumalkenivorans AK-01]; gi|220909520|ref|YP 002484831.1| acetolactatedecarboxylase [Cyanothece sp. PCC 7425]; gi|218782031|ref|YP002433349.1| acetolactate decarboxylase [Desulfatibacillum alkenivoransAK-01]; gi|213693090|ref|YP_002323676.11 acetolactate decarboxylase[Bifidobacterium longum subsp. infantis ATCC 15697=JCM 1222];gi|189500297|ref|YP 001959767.1| acetolactate decarboxylase [Chlorobiumphaeobacteroides BS1]; gi|189423787|ref|YP 001950964.11 acetolactatedecarboxylase [Geobacter lovleyi SZ]; gi|172058271|ref|YP 001814731.11acetolactate decarboxylase [Exiguobacterium sibiricum 255-15];gi|163938775|ref|YP 001643659.11 acetolactate decarboxylase [Bacillusweihenstephanensis KBAB4]; gi|158522304|ref|YP 001530174.11 acetolactatedecarboxylase [Desulfococcus oleovorans Hxd3]; gi|57371670|ref|YP001479659.11 acetolactate decarboxylase [Serratia proteamaculans 568];gi|150395111|ref|YP 001317786.11 acetolactate decarboxylase[Staphylococcus aureus subsp. aureus JH1]; gi|150394715|ref|YP001317390.11 acetolactate decarboxylase [Staphylococcus aureus subsp.aureus JH1]; gi|146311679|ref|YP 001176753.1| acetolactate decarboxylase[Enterobacter sp. 638]; gi|109900061|ref|YP 663316.11 acetolactatedecarboxylase [Pseudoalteromonas atlantica T6c];gi|219866131|gb|ACL46470.1| acetolactate decarboxylase [Cyanothece sp.PCC 7425]; gi|213524551|gb|ACJ53298.1|acetolactate decarboxylase[Bifidobacterium longum subsp. infantis ATCC 15697=JCM 1222];gi|189420046|gb|ACD94444.1| acetolactate decarboxylase [Geobacterlovleyi SZ]; gi|158511130|gb|ABW68097.1|acetolactate decarboxylase[Desulfococcus oleovorans Hxd3]; gi|157323434|gb|ABV42531.1|acetolactatedecarboxylase [Serratia proteamaculans 568];gi|145318555|gb|ABP60702.1|acetolactate decarboxylase [Enterobacter sp.638]; gi|149947563|gb|ABR53499.1|acetolactate decarboxylase[Staphylococcus aureus subsp. aureus JH1];gi|149947167|gb|ABR53103.1|acetolactate decarboxylase[Staphylococcusaureus subsp. aureus JH1];gi|163860972|gb|ABY42031.1|Acetolactate decarboxylase [Bacillusweihenstephanensis KBAB4]; gi|109702342|gb|ABG42262.1|Acetolactatedecarboxylase [Pseudoalteromonas atlantica T6c];gi|189495738|gb|ACE04286.1|acetolactate decarboxylase [Chlorobiumphaeobacteroides BS1]; gi|171990792|gb|ACB61714.1|acetolactatedecarboxylase [Exiguobacterium sibiricum 255-15]; gi|223932563|ref|ZP03624564.1 acetolactate decarboxylase [Streptococcus suis 89/1591];gi|194467531|ref|ZP 03073518.11 acetolactate decarboxylase[Lactobacillus reuteri 100-23]; gi|223898834|gb|EEF65194.1|acetolactatedecarboxylase [Streptococcus suis 89/1591];gi|194454567|gb|EDX43464.1|acetolactate decarboxylase [Lactobacillusreuteri 100-23]; gi|384267135|ref|YP 005422842.11 acetolactatedecarboxylase [Bacillus amyloliquefaciens subsp. plantarum YAUB9601-Y2]; gi|375364037|ref YP_005132076.11 acetolactate decarboxylase[Bacillus amyloliquefaciens subsp. plantarum CAU B946];gi|34079323|ref|YP_004758694.11 acetolactate decarboxylase[Corynebacterium variabile DSM 44702]; gi|336325119|ref|YP_004605085.11acetolactate decarboxylase [Corynebacterium resistens DSM 45100];gi|148269032|ref|YP_001247975.1| acetolactate decarboxylase[Staphylococcus aureus subsp. aureus JH9];gi|148268650|ref|YP_001247593.11 acetolactate decarboxylase[Staphylococcus aureus subsp. aureus JH9];gi|1485433721|ref|YP_001270742.11 acetolactate decarboxylase[Lactobacillus reuteri DSM 20016];gi|380500488|emb|CCG51526.1|acetolactate decarboxylase [Bacillusamyloliquefaciens subsp. plantarum YAU B9601-Y2];gi|371570031|emb|CCF06881.1| acetolactate decarboxylase [Bacillusamyloliquefaciens subsp. plantarum CAU B946];gi|340533141|gb|AEK35621.1|acetolactate decarboxylase [Corynebacteriumvariabile DSM 44702]; gi|336101101|gb|AE108921.1|acetolactatedecarboxylase [Corynebacterium resistens DSM 45100];gi|148530406|gb|ABQ82405.1|acetolactate decarboxylase [Lactobacillusreuteri DSM 20016]; gi|147742101|gb|ABQ50399.1|acetolactatedecarboxylase [Staphylococcus aureus subsp. aureus JH9];gi|147741719|gb|ABQ50017.1|acetolactate decarboxylase [Staphylococcusaureus subsp. aureus JH9]; gi|392529510|ref|ZP 10276647.11 acetolactatedecarboxylase [Carnobacterium maltaromaticum ATCC 35586];gi|366054074|ref|ZP 09451796.11 acetolactate decarboxylase[Lactobacillus suebicus KCTC 3549]; gi|339624147|ref|ZP 08659936.11acetolactate decarboxylase [Fructobacillus jructosus KCTC 3544]; andgi|336393727|ref|ZP 08575126.11 acetolactate decarboxylase[Lactobacillus coryniformis subsp. torquens KCTC 3535]. UNIPROTAccession No. P23616.1 (Diderichsen et al., J Bacteriol. (1990) 172(8):4315) is an example of an ALDC enzyme. UNIPROT accession number P23616.1is an example of an ALDC enzyme derived or derivable from Bacillusbrevis. Each sequence associated with the foregoing accession numbers isincorporated herein by reference.

In some embodiments, the invention relates to ALDC enzymes fromLactobacillus casei (Godtfredsen 1984), Brevibacterium acetylicum(Oshiro, 1989), Lactococcus lactis (Vincent Phalip 1994), Leuconostoclactis (0 sulivan, 2001), Enterobacter aerogenes (Blomquist, 1993),Bacillus subtilis (Renna, 1993), Bacillus brevis (Svendsen, 1989) andLactococcus lactis DX (Yuxing, 2014). In some embodiments, the ALDCenzyme is from Lactobacillus casei, Brevibacterium acetylicum,Lactococcus lactis, Leuconostoc lactis, Enterobacter aerogenes, Bacillussubtilis, Bacillus brevis, Lactococcus lactis DX, or Bacilluslicheniformis. As used herein, the term “ALDC enzyme is from” refers tothe ALDC enzyme being derived or derivable from.

It is to be understood that any suitable ALDC enzymes, i.e. ALDCproduced from any microorganism which activity is dependent on metalions, can be used according to the invention. In some embodiments, theALDC used in the methods and compositions described herein is an ALDCfrom Bacillus brevis or Bacillus licheniformis.

The ALDC activity of the enzyme composition according to the inventionis measured by the ALDC assays as described herein or any suitable assayknown in the art. The standard assay is carried out at pH 6.0, and itcan be performed at different pH values and temperatures for theadditional characterization and specification of enzymes.

One unit of ALDC activity is defined as the amount of enzyme whichproduces 1 μmole acetoin per minute under the conditions of the assay(e.g., pH 6.0 (or as specified) and 30° C.).

In some embodiments, the ALDC is an ALDC derivative. In someembodiments, the ALDC derivative is characterized by the fact that ALDCin an aqueous medium is treated with or has been treated withglutaraldehyde. In some embodiments, the ALDC is treated with or hasbeen treated with glutaraldehyde in a concentration corresponding tobetween 0.1 and 5 g of glutaraldehyde per g of pure ALDC protein,preferably corresponding to between 0.25 and 2 g of glutaraldehyde per gof pure ALDC protein.

In some embodiments, the ALDC enzyme comprises an amino acid sequencehaving at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identity with SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, orSEQ ID NO: 8 or any functional fragment thereof. One aspect of theinvention relates to an enzyme exhibiting ALDC activity, which enzymecomprises an amino acid sequence having at least 80% identity with anyone selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO:7, SEQ ID NO: 8 or any functional fragment thereof. In some embodiments,the ALDC enzyme is encoded by a nucleic acid sequence having at least70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identity with SEQ IDNO: 1, SEQ ID NO: 4, or SEQ ID NO: 6 or any functional fragment thereof.

In some embodiments, the enzyme has a temperature optimum in the rangeof 5-80° C., such as in the range of 5-40° C. or 15-80° C., such as inthe range 20-80° C., such as in the range 5-15° C., 10-40° C., 10-50°C., 15-20° C., 45-65° C., 50-65° C., 55-65° C. or 60-80° C. In someembodiments, the enzyme has a temperature optimum of about 60° C.

In some embodiments, the enzyme has a total number of amino acids ofless than 350, such as less than 340, such as less than 330, such asless than 320, such as less than 310, such as less than 300 amino acids,such as in the range of 200 to 350, such as in the range of 220 to 345amino acids.

In some embodiments, the amino acid sequence of the enzyme has at leastone, two, three, four, five, six, seven, eight, nine or ten amino acidsubstitutions as compared to any one amino acid sequence selected fromSEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, orany functional fragment thereof.

In some embodiments, the amino acid sequence of the enzyme has a maximumof one, two, three, four, five, six, seven, eight, nine or ten aminoacid substitutions compared to any one amino acid sequence selected fromSEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, orany functional fragment thereof.

In some embodiments, the enzyme comprises the amino acid sequenceidentified by any one of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQID NO: 7, or SEQ ID NO: 8, or any functional fragment thereof.

In some embodiments the compositions, media and methods according to theinvention comprise any one or more further enzyme. In some embodimentsthe one or more further enzyme is selected from list consisting ofacetolactate reductoisomerases, acetolactate isomerases, amylase,glucoamylase, hemicellulase, cellulase, glucanase, pullulanase,isoamylase, endo-glucanase and related beta-glucan hydrolytic accessoryenzymes, xylanase, xylanase accessory enzymes (for example,arabinofuranosidase, ferulic acid esterase, xylan acetyl esterase) andprotease.

In some embodiments the compositions, media and methods according to theinvention comprise an enzyme exhibiting ALDC activity, wherein theactivity of said ALDC enzyme is in the range of 950 to 2500 Units per mgof protein. In some embodiments the compositions, media and methodsaccording to the invention comprise an enzyme exhibiting ALDC activity,wherein the activity of said ALDC enzyme is in the range of 1000 to 2500Units per mg of protein. In some embodiments the compositions, media andmethods according to the invention comprise an enzyme exhibiting ALDCactivity, wherein the activity of said ALDC enzyme is in the range of1500 to 2500 Units per mg of protein. In some embodiments, the enzymeexhibiting ALDC activity is an enzyme comprising an amino acid sequencehaving at least 80% identity with any one selected from SEQ ID NO: 2,SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, or anyfunctional fragment thereof. In some embodiments, the enzyme exhibitingALDC activity is encoded by a nucleic acid sequence having at least 80%identity with SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 6 or any functionalfragment thereof.

Metal Ions

In one aspect, the invention provides methods and compositionscomprising ALDC enzymes having a better stability and/or activity. Inanother aspect the invention provides methods and compositionscomprising ALDC enzymes which can be recovered from microorganisms inimproved yields.

Surprisingly, it has been found by the present inventors that treatmentof ALDC compositions with certain metal ions at certain concentrationsprovides ALDC enzymes having a better stability and/or activity, and,optionally, the yield of ALDC activity which can be recovered frommicroorganisms is improved.

In some embodiments, the atomic radius for the metal ion is about 140 pmto about 255 pm. In some embodiments, the atomic radius for the metalion is about 140 pm to about 165 pm. In some embodiments, the atomicradius for the metal ion is about 140 pm to about 150 pm. In someembodiments, the atomic radius for the metal ion is about 142 pm toabout 146 pm.

In some embodiments, the metal ion is selected from the group consistingof Zn²⁺, Mg²⁺, Mn²⁺, Co²⁺, Cu²⁺, Ba²⁺, Ca²⁺ and Fe²⁺ and combinationsthereof. In some embodiments, the metal ion is selected from the groupconsisting of Zn²⁺, Cu²⁺, and Fe²⁺. In some embodiments, the metal ionis selected from the group consisting of Zn²⁺, Mn²⁺, and Co²⁺. In someembodiments, the metal ion is Zn²⁺ or Mn²⁺. In some embodiments, themetal ion is Zn²⁺. The term “zinc” as used herein may be interchangeablewith the term “Zn²⁺”. The term “metal” as used herein may beinterchangeable with the term “metal ion”. The term “metal” as usedherein may refer to compounds which comprise the metal selected from thegroup consisting of zinc, magnesium, manganese, cobalt, copper, barium,calcium and iron; compounds which comprise these metals are a source ofthe respective ions. The term “zinc” as used herein refers to compoundswhich comprise zinc, such compounds are a source of Zn²⁺ ions. Zincsulfate (ZnSO₄) is example of zinc as referred to herein and is anexample of a source of Zn²⁺ ions. Magnesium sulfate (MgSO₄) is anexample of magnesium as referred to herein and is an example of a sourceof Mg²⁺ ions. Manganese(II) sulfate (MnSO₄) is an example of manganeseas referred to herein and is an example of a source of Mn²⁺ ions.Cobalt(II)chloride (CoCl₂) is an example of cobalt as referred to hereinand is an example of a source of Co²⁺ ions. Copper(II) sulphate (CuSO₄)is an example of copper as referred to herein and is an example of asource of Cu²⁺ ions. Barium sulfate (BaSO₄) is an example of barium asreferred to herein and is an example of a source of Ba²⁺ ions. Calciumsulfate (CaSO₄) is an example of calcium as referred to herein and isexample of a source of Ca²⁺ ions. Iron(II) sulfate (FeSO₄) is an exampleof iron as referred to herein and is example of a source of Fe²⁺ ions.

The present inventors have found that metal ions such as Zn²⁺, Mn²⁺,Co²⁺, Cu²⁺, and Fe²⁺ increase the stability of ALDC enzyme in differentformulations (see Examples), and also improve the recovery yields frommicroorganisms when the metal ions are used during the production of theenzyme in the cultivation media. Thus, in some embodiments, the presentinvention provides methods and compositions that increase the recoveryyields, stability and/or activity of ALDC enzymes that can be then used,e.g., to produce fermented products such as in brewing.

In one aspect, the present disclosure provides compositions comprisingan ALDC enzyme with increased stability and/or activity.

In some embodiments, the ALDC enzyme has an specific activity of atleast about 900 units per mg of protein (U/mg), at least about 1000U/mg, at least about 1500 U/mg, at least about 2000 U/mg, at least about3000 U/mg at least about 5000 U/mg, at least about 6000 U/mg, at leastabout 7000 U/mg, at least about 8000 U/mg, at least about 8500 U/mg, atleast about 9000 U/mg, at least about 9500 U/mg, or at least about 10000U/mg as measured by the assays described herein or any suitable assayknown in the art. In some embodiments, the ALDC enzyme has an ALDCactivity in the range of about 950 to 2500 units per mg of protein(U/mg), about 1000 to 2500 U/mg, or about 1500 to 2500 U/mg as measuredby the assays described herein or any suitable assay known in the art.In some embodiments, the ALDC compositions according to the inventioncomprise an ALDC enzyme with ALDC activity of at least about 900 unitsper gram of product, at least about 1000 U/g, at least about 1500 U/g,at least about 2000 U/g, at least about 3000 U/g at least about 5000U/g, such as at least about 6000 U/g, such as at least about 7000 U/g,such as at least about 8000 U/g, such as at least about 8500 U/g, suchas at least about 9000 U/g, such as at least about 9500 U/g, such as atleast about 10000 U/g as measured by in the assays described herein orany suitable assay known in the art. In some embodiments, a differentALDC activity is used, e.g., depending on the acetolactate content andconditions requirements, e.g. for brewing. In some embodiments, the ALDCcompositions according to the invention comprise an ALDC enzyme withALDC activity of at least about 8000 U/g.

In some embodiments, the compositions comprise an ALDC enzyme and ametal ion, where the metal ion is present at a concentration of about0.1 μM to about 200 mM, such as about 1 μM to about 200 mM, or about 1μM to about 500 μM, or about 1 μM to about 300 μM, or about 6 μM toabout 300 μM, or about 10 μM to about 100 μM, or about 15 μM to about 50μM, or about 1 μM to about 150 mM, or about 10 μM to about 150 mM, orabout 20 μM to about 120 mM, or about 25 μM to about 100 mM, or about 25μM to about 50 mM, or about 25 μM to about 20 mM, or about 25 μM toabout 50 μM, or about 100 μM to about 20 mM, or about 250 μM to about 20mM, or about 1 mM to about 20 mM, or about 1 mM to about 5 mM. In someembodiments, the compositions comprise an ALDC enzyme and a metal ion,where the metal ion is present at a concentration of about 1 μM to about300 μM, such as about 6 μM to about 300 μM, or about 6 μM to about 50μM, or about 6 μM to about 25 μM. In some embodiments, the compositionscomprise an ALDC enzyme and a metal ion, where the metal ion is presentat a concentration of about 60 μM to about 150 μM, or about 25 μM toabout 150 μM. In some embodiments, the compositions comprise an ALDCenzyme and a metal ion, where the metal ion is present at aconcentration of about 100 μM to about 200 mM. In some embodiments, thecompositions comprise an ALDC enzyme and a metal ion, where the metalion is present at a concentration of about 100 μM to about 20 mM. Insome embodiments, the compositions comprise an ALDC enzyme and a metalion, where the metal ion is present at a concentration of about 1 mM toabout 5 mM. In some embodiments, the metal ion is selected from thegroup consisting of Zn²⁺, Mg²⁺, Mn²⁺, Co²⁺, Cu²⁺, Ba²⁺, Ca²⁺ and Fe²⁺and combinations thereof. In some embodiments, the metal ion is selectedfrom the group consisting of Zn²⁺, Cu²⁺, and Fe²⁺. In some embodiments,the metal ion is selected from the group consisting of Zn²⁺, Mn²⁺, andCo²⁺. In some embodiments, the metal ion is Zn²⁺ or Mn²⁺. In someembodiments, the metal ion is Zn²⁺.

In some embodiments, the compositions comprise an ALDC enzyme and zincwhere the zinc is present at a concentration of about 1 μM to about 200mM, such as about 1 μM to about 500 μM, or about 1 μM to about 300 μM,or about 6 μM to about 300 μM, or about 10 μM to about 100 μM, or about15 μM to about 50 μM, or about 10 μM to about 150 mM, or about 20 μM toabout 120 mM, or about 25 μM to about 100 mM, or about 25 μM to about 50mM, or about 25 μM to about 20 mM, or about 25 μM to about 50 μM, orabout 100 μM to about 20 mM, or about 250 μM to about 20 mM, or about500 μM to about 20 mM, or about 1 mM to about 20 mM, or about 1 mM toabout 10 mM, or about 1 mM to about 5 mM, or about 5 mM to about 20 mM,or about 5 mM to about 10 mM. In some embodiments, the compositionscomprise an ALDC enzyme and zinc, where the zinc is present at aconcentration of about 1 μM to about 300 μM, such about 6 μM to about300 μM, or about 6 μM to about 25 μM. In some embodiments, thecompositions comprise an ALDC enzyme and zinc, where the zinc is presentat a concentration of about 25 μM to about 150 μM or about 60 μM toabout 150 μM. In some embodiments, the compositions comprise an ALDCenzyme and zinc, where the zinc is present at a concentration of about100 μM to about 20 mM. In some embodiments, the compositions comprise anALDC enzyme and zinc, where the zinc is present at a concentration ofabout 100 μM to about 10 mM. In some embodiments, the compositionscomprise an ALDC enzyme and zinc, where the zinc is present at aconcentration of about 1 mM to about 5 mM.

In some embodiments, the compositions comprise an ALDC enzyme and zincwhere the zinc is present at a concentration of about 1 mM to about 3mM, or about 0.75 mM to about 4 mM, or about 0.5 mM to about 5 mM, orabout 0.25 mM to about 7.5 mM, or about 0.1 mM to about 10 mM. In someembodiments, the activity of said ALDC enzyme is in the range of 950 to2500 Units per mg of protein, or 1000 to 2500 Units per mg of protein,or 1500 to 2500 Units per mg of protein.

In some embodiments, the compositions comprise an ALDC enzyme and zinc,where the molar ratio of zinc to enzyme is higher than 1 such as 2:1, or3:1, or 5:1, or 10:1, or 20:1 or 30:1, or 50:1, or 60:1, or 100:1, or150:1, or 200:1, or 250:1, or 500:1. In some embodiments, thecompositions comprise an ALDC enzyme and zinc, where the molar ratio ofzinc to enzyme is 2:1 or higher. In some embodiments, the compositionscomprise an ALDC enzyme and zinc, where the molar ratio of zinc toenzyme is 5:1 or higher. In some embodiments, the compositions comprisean ALDC enzyme and zinc, where the molar ratio of zinc to enzyme is 10:1or higher. In some embodiments, the compositions comprise an ALDC enzymeand zinc, where the molar ratio of zinc to enzyme is 20:1 or higher. Insome embodiments, the compositions comprise an ALDC enzyme and zinc,where the molar ratio of zinc to enzyme is 30:1 or higher. In someembodiments, the compositions comprise an ALDC enzyme and zinc, wherethe molar ratio of zinc to enzyme is 60:1 or higher. The molarconcentration of, for example, Zn²⁺, Mn²⁺, Co²⁺ or other metal ions insolution may be determined by inductively coupled plasma opticalemission spectrometry (ICP-OES) or similar techniques. The molarconcentration of the ALDC enzyme may be determined using. CriterionSLS-PAGE system (such as described in the examples) and the amino acidsequence.

In some embodiments, the ALDC enzyme is an ALDC derivative. In someembodiments, the ALDC derivative is an ALDC enzyme treated withglutaraldehyde. In some embodiments, the ALDC enzyme is treated withglutaraldehyde at a concentration corresponding to about 0.1 to about 5g of glutaraldehyde per g of pure ALDC enzyme.

In some embodiments, the activity of the ALDC enzyme is in the range of950 to 2500 Units per mg of protein. In some embodiments, the activityof said ALDC enzyme is in the range of 1000 to 2500 Units per mg ofprotein. In some embodiments, the activity of said ALDC enzyme is in therange of 1500 to 2500 Units per mg of protein. Thus, in someembodiments, the compositions comprise an ALDC enzyme, where the ALDCenzyme is in the range of 950 to 2500 Units per mg of protein or 1000 to2500 Units per mg of protein or 1500 to 2500 Units per mg of protein.

In some embodiments, the compositions comprise an ALDC enzyme and zinc,where the zinc is present at a concentration of about 100 μM to about 20mM, and the activity of said ALDC enzyme is in the range of 1000 to 2500Units per mg of protein. In some embodiments, the compositions comprisean ALDC enzyme or an ALDC derivative and zinc, where the zinc is presentat a concentration of about 250 μM to about 20 mM, and the activity ofsaid ALDC enzyme is in the range of 1000 to 2500 Units per mg ofprotein.

In some embodiments, the ALDC enzyme compositions further comprise atleast one additional enzyme or enzyme derivative selected from the groupconsisting of acetolactate reductoisomerases, acetolactate isomerases,amylase, glucoamylase, hemicellulase, cellulase, glucanase, pullulanase,isoamylase, endo-glucanase and related beta-glucan hydrolytic accessoryenzymes, xylanase, xylanase accessory enzymes (for example,arabinofuranosidase, ferulic acid esterase, and xylan acetyl esterase)and protease.

In some embodiments, the ALDC enzyme compositions described herein areused during fermentation and/or maturation of a beverage preparationprocess, e.g., beer and wine, to reduce diacetyl levels. The terms “ALDCenzyme composition”, “composition comprising an ALDC enzyme” and“composition comprising ALDC” as used herein refer to compositionscomprising the ALDC enzyme. The composition may be in the form of asolution. As used herein, the terms “ALDC enzyme composition” and“compositions comprising ALDC” are mutually exclusive with media (suchas cultivation media, fermentation media or maturation media) whichcomprise microorganisms expressing ALDC and/or capable of expressingALDC when cultured under conditions permitting expression of the enzyme.Examples of ALDC enzyme compositions and compositions comprising ALDCinclude compositions comprising ALDC in a purified form. ALDC may bepurified from a media comprising microorganisms capable of expressingALDC wherein said media has been cultured under conditions permittingexpression of ALDC. The term “purified” means that ALDC is present at ahigh level. Preferably, ALDC is the predominant component present in thecomposition. Preferably, ALDC is present at a level of at least about90%, or at least about 95% or at least about 98%, said level beingdetermined on a dry weight/dry weight basis with respect to the totalcomposition under consideration. In some embodiments, an ALDC enzymecomposition further comprises a metal ion such as zinc.

As used herein, the terms “beverage” and “beverage(s) product” includesuch foam forming fermented beverages as beer brewed with 100% malt,beer brewed under different types of regulations, ale, dry beer, nearbeer, light beer, low alcohol beer, low calorie beer, porter, bock beer,stout, malt liquor, non-alcoholic beer, non-alcoholic malt liquor andthe like. The term “beverages” or “beverages product” also includesnon-foaming beer and alternative malt beverages such as fruit flavoredmalt beverages, e. g., citrus flavored, such as lemon-, orange-, lime-,or berry-flavored malt beverages, liquor flavored malt beverages, e. g.,vodka-, rum-, or tequila-flavored malt liquor, or coffee flavored maltbeverages, such as caffeine-flavored malt liquor, and the like. The term“beverages” or “beverages product” also includes beer made withalternative materials other than malted barley, such as rye, corn, oats,rice, millet, triticale, cassava, sorghum, barley, wheat and acombination thereof. The term “beverages” or “beverages product” alsoincludes other fermented products such as wine or ciders or perry orsake.

Beer is traditionally referred to as an alcoholic beverage derived frommalt, such as malt derived from barley grain, and optionally adjunct,such as starch containing plant material (e.g. cereal grains) andoptionally flavored, e.g. with hops. In the context of the presentinvention, the term “beer” includes any fermented wort, produced byfermentation/brewing of a starch-containing plant material, thus inparticular also beer produced exclusively from adjunct, or anycombination of malt and adjunct. Beer can be made from a variety ofstarch-containing plant material by essentially the same process, wherethe starch consists mainly of glucose homopolymers in which the glucoseresidues are linked by alpha-1, 4- or alpha-1,6-bonds, with the formerpredominating. Beer can be made from alternative materials such as rye,corn, oats, rice, millet, triticale, cassava, sorghum, wheat, barley anda combination thereof.

In some embodiments, the invention provides a fermentation media (e.g.beer and/or wine and/or cider and/or perry and/or sake fermentation)comprising an ALDC enzyme and metal ion at a concentration of about 0.1μM to about 200 mM, or about 1 μM to about 200 mM, such as about 1 μM toabout 500 μM, or about 0.1 μM to about 300 μM, or about 1 μM to about300 μM, or about 6 μM to about 300 μM, or about 1 μM to about 100 μM, orabout 1 μM to about 50 μM, or about 6 μM to about 50 μM, or about 6 μMto about 25 μM. In some embodiments, the invention provides afermentation media (e.g. beer and/or wine and/or cider and/or perryand/or sake fermentation) comprising an ALDC enzyme and metal ion at aconcentration of about 0.1 μM to about 100 mM, such as about 0.1 μM toabout 10 μM, or 1 μM to about 100 mM, or 1 μM to about 10 μM, or 6 μM toabout 10 μM, or about 10 μM to about 200 μM, or about 50 μM to about 1mM, or about 100 μM to about 10 mM, or about 100 μM to about 50 mM, orabout 100 μM to about 100 mM, or about 100 μM to about 200 mM, or about250 μM to about 120 mM, or about 500 μM to about 100 mM, or about 1 mMto about 50 mM, or about 1 mM to about 20 mM, or about 1 mM to about 5mM. In some embodiments, the invention provides a fermentation media(e.g. beer and/or wine and/or cider and/or perry and/or sakefermentation) comprising an ALDC enzyme and metal ion at a concentrationof about 0.1 μM to about 200 mM or about 1 μM to about 200 mM, such asabout 1 μM to about 500 μM, or about 1 μM to about 300 μM, or about 6 μMto about 300 μM, or about 1 μM to about 100 μM, or about 1 μM to about50 μM, or about 6 μM to about 50 μM, or about 6 μM to about 25 μM. Insome embodiments, the invention provides a fermentation media (e.g. beerand/or wine and/or cider and/or perry and/or sake fermentation)comprising an ALDC enzyme and metal ion at a concentration of about 1 μMto about 300 μM, or about 6 μM to about 300 μM, or about 1 μM to about100 μM, or about 1 μM to about 50 μM, or about 6 μM to about 50 μM orabout 6 μM to about 25 μM. In some embodiments, the metal ion isselected from the group consisting of Zn²⁺, Mg²⁺, Mn²⁺, Co²⁺, Cu²⁺,Ba²⁺, Ca²⁺ and Fe²⁺ and combinations thereof. In some embodiments, themetal ion is selected from the group consisting of Zn²⁺, Cu²⁺, and Fe²⁺.In some embodiments, the metal ion is selected from the group consistingof Zn²⁺, Mn²⁺, and Co²⁺. In some embodiments, the metal ion is Zn²⁺ orMn²⁺. In some embodiments, the metal ion is Zn²⁺. In some embodiments,the activity of said ALDC enzyme is in the range of 950 to 2500 Unitsper mg of protein, or 1000 to 2500 Units per mg of protein, or 1500 to2500 Units per mg of protein. In some embodiments, the fermentationmedia (e.g. beer and/or wine and/or cider and/or perry and/or sakefermentation) further comprises at least one additional enzyme or enzymederivative selected from the group consisting of acetolactatereductoisomerases, acetolactate isomerases, amylase, glucoamylase,hemicellulase, cellulase, glucanase, pullulanase, isoamylase,endo-glucanase and related beta-glucan hydrolytic accessory enzymes,xylanase, xylanase accessory enzymes (for example, arabinofuranosidase,ferulic acid esterase, and xylan acetyl esterase) and protease.

In some embodiments, the invention provides a maturation media (e.g.beer and/or wine and/or cider and/or perry and/or sake fermentation)comprising an ALDC enzyme and metal ion at a concentration of about 0.1μM to about 200 mM, or 1 μM to about 200 mM, such as about 1 μM to about500 μM, or about 0.1 μM to about 300 μM, or about 1 μM to about 300 μM,or about 6 μM to about 300 μM, or about 1 μM to about 100 μM, or about 1μM to about 50 μM, or about 6 μM to about 50 μM, or about 6 μM to about25 μM. In some embodiments, the invention provides a maturation media(e.g. beer and/or wine and/or cider and/or perry and/or sakefermentation) comprising an ALDC enzyme and metal ion at a concentrationof about 0.1 μM to about 100 mM, or 1 μM to about 100 mM, such as about0.1 μM to about 10 μM, or 1 μM to about 10 μM, or 6 μM to about 10 μM,or about 10 μM to about 200 μM, or about 50 μM to about 1 mM, or about100 μM to about 10 mM, or about 100 μM to about 50 mM, or about 100 μMto about 100 mM, or about 100 μM to about 200 mM, or about 250 μM toabout 120 mM, or about 500 μM to about 100 mM, or about 1 mM to about 50mM, or about 1 mM to about 20 mM, or about 1 mM to about 5 mM. In someembodiments, the invention provides a maturation media (e.g. beer and/orwine and/or cider and/or perry and/or sake fermentation) comprising anALDC enzyme and metal ion at a concentration of about 1 μM to about 500μM, or about 1 μM to about 300 μM, or about 6 μM to about 300 μM, orabout 1 μM to about 100 μM, or about 1 μM to about 50 μM, or about 6 μMto about 50 μM, or about 6 μM to about 25 μM. In some embodiments, theinvention provides a maturation media (e.g. beer and/or wine and/orcider and/or perry and/or sake fermentation) comprising an ALDC enzymeand metal ion at a concentration of about 1 μM to about 300 μM, or about6 μM to about 300 μM, or about 1 μM to about 100 μM, or about 1 μM toabout 50 μM, or about 6 μM to about 50 μM, or about 6 μM to about 25 μM.In some embodiments, the metal ion is selected from the group consistingof Zn²⁺, Mg²⁺, Mn²⁺, Co²⁺, Cu²⁺, Ba²⁺, Ca²⁺ and Fe²⁺ and combinationsthereof. In some embodiments, the metal ion is selected from the groupconsisting of Zn²⁺, Cu²⁺, and Fe²⁺. In some embodiments, the metal ionis selected from the group consisting of Zn²⁺, Mn²⁺, and Co²⁺. In someembodiments, the metal ion is Zn²⁺ or Mn²⁺. In some embodiments, themetal ion is Zn²⁺. In some embodiments, the activity of said ALDC enzymeis in the range of 950 to 2500 Units per mg of protein, or 1000 to 2500Units per mg of protein, or 1500 to 2500 Units per mg of protein. Insome embodiments, the maturation media (e.g. beer and/or winematuration) further comprises at least one additional enzyme or enzymederivative selected from the group consisting of acetolactatereductoisomerases, acetolactate isomerases, amylase, glucoamylase,hemicellulase, cellulase, glucanase, pullulanase, isoamylase,endo-glucanase and related beta-glucan hydrolytic accessory enzymes,xylanase, xylanase accessory enzymes (for example, arabinofuranosidase,ferulic acid esterase, and xylan acetyl esterase) and protease.

In some embodiments, metal ions such as Zn²⁺, Mg²⁺, Mn²⁺, Co²⁺, Cu²⁺,Ba²⁺, Ca²⁺ and Fe²⁺ and combinations thereof are added to thecultivation and/or fermentation media during and/or after ALDCproduction to increase the recovered yields from microorganisms.

The term “cultivation media” as used herein refers to a media whichsupports the growth of microorganisms such as an ALDC producing hostcell. Examples of a cultivation media include: media based on MOPsbuffer with, for instance, urea as the major nitrogen source and maltrinas the main carbon source; and TSB broth. In some embodiments, theinvention provides a cultivation media for an ALDC producing host cellcomprising a metal ion at a concentration of about 1 μM to about 1 mM.In some embodiments, the invention provides a cultivation media for anALDC producing host cell comprising a metal ion at a concentration ofabout 25 μM to about 150 μM. In some embodiments, the invention providesa cultivation media for an ALDC producing host cell comprising a metalion at a concentration of about 25 μM to about 50 μM. In someembodiments, the invention provides a cultivation media for an ALDCproducing host cell comprising a metal ion at a concentration of about30 μM to about 40 μM. In some embodiments, the invention provides acultivation media for an ALDC producing host cell comprising a metal ionat a concentration of about 40 μM to about 150 μM. In some embodiments,the invention provides a cultivation media for an ALDC producing hostcell comprising a metal ion at a concentration of about 60 μM to about150 μM. In some embodiments, the metal ion is selected from the groupconsisting of Zn2+, Mg²⁺, Mn²⁺, Co²⁺, Cu²⁺, Ba²⁺, Ca²⁺ and Fe²⁺ andcombinations thereof. In some embodiments, the metal ion is selectedfrom the group consisting of Zn²⁺, Cu²⁺, and Fe²⁺. In some embodiments,the metal ion is selected from the group consisting of Zn²⁺, Mn²⁺, andCo²⁺. In some embodiments, the metal ion is Zn²⁺ or Mn²⁺. In someembodiments, the metal ion is Zn²⁺. In some embodiments, the activity ofsaid ALDC enzyme is in the range of 950 to 2500 Units per mg of protein,or 1000 to 2500 Units per mg of protein, or 1500 to 2500 Units per mg ofprotein.

Materials may be added to an enzyme-containing composition to improvethe properties of the composition. Non-limiting examples of suchadditives include: salts (e.g., alkali salts, earth metal salts,additional chloride salts, sulfate salts, nitrate salts, carbonatesalts, where exemplary counter ions are calcium, potassium, and sodium),inorganic minerals or clays (e.g., zeolites, kaolin, bentonite, talcsand/or silicates), carbohydrates (e.g., sucrose and/or starch), coloringpigments (e.g., titanium dioxide), biocides (e.g., Rodalon®, Proxel®),dispersants, anti-foaming agents, reducing agents, acid agents, alkalineagents, enzyme stabilizers (e.g. polyol such as glycerol, propyleneglycol, sorbitol, inorganic salts, sugars, sugar or a sugar alcohol,lactic acid, boric acid, or a boric acid derivative and combinationsthereof), enzyme inhibitors, preservative (e.g. methyl paraben, propylparaben, benzoate, sorbate or other food approved preservatives) andcombinations thereof. Excipients which may be used in the composition,or the preparation thereof, include maltose, maltose syrup, sucrose,glucose (including glucose syrup or dried glucose syrup), pre-cookedstarch, gelatinised starch, L-lactic, ascorbyl palmitate, tocopherols,lecithins, citric acid, citrates, phosphoric, phosphates, sodiumalginate, carrageenan, locust bean gum, guar gum, xanthan gum, pectins,sodium carboxymethylcellulose, mono- and diglycerides, citric acidesters of mono- and diglycerides, sucrose esters, carbon dioxide, argon,helium, nitrogen, nitrous oxide, oxygen, hydrogen, and starch sodiumoctenylsuccinate.

Methods

In some aspects the invention provides methods to improve stabilityand/or activity of ALDC enzymes. In some aspects the invention providesmethods to improve ALDC recovery from microorganisms.

In some embodiments, the invention provides methods for increasing theactivity and/or stability of an ALDC enzyme in a composition comprisingALDC wherein said method comprises the step of adding a metal ion to thecomposition so that said metal ion is present in said composition at aconcentration of about 1 μM to about 200 mM, such as about 1 μM to about500 μM, or about 1 μM to about 300 μM, or about 6 μM to about 300 μM, orabout 1 μM to about 100 μM, or about 1 μM to about 50 μM, or about 10 μMto about 150 mM, or about 20 μM to about 120 mM, or about 25 μM to about100 mM, or about 25 μM to about 50 mM, or about 25 μM to about 20 mM, orabout 25 μM to about 50 μM, or about 100 μM to about 20 mM, or about 250μM to about 20 mM, or about 500 μM to about 20 mM, or about 1 mM toabout 20 mM, or about 1 mM to about 10 mM, or about 1 mM to about 5 mM,or about 5 mM to about 20 mM, or about 5 mM to about 10 mM. In someembodiments, the invention provides methods for increasing the activityand/or stability of an ALDC enzyme in a cultivation media comprising anALDC producing host cell wherein said method comprises the step ofadding a metal ion to the media so that said metal ion is present insaid media at a concentration of about 1 μM to about 1 mM, such as about1 μM to about 300 μM, about 6 μM to about 300 μM, about 25 μM to about150 μM, or about 60 μM to about 150 μM. In some embodiments, theinvention provides methods for increasing the activity and/or stabilityof an ALDC enzyme in a fermentation and/or maturation media comprisingan ALDC enzyme wherein said method comprises the step of adding a metalion to the media so that said metal ion is present in said media at aconcentration of about 1 μM to about 300 μM, such as about 6 μM to about300 μM, about 1 μM to about 100 μM, about 1 μM to about 50 μM, about 1μM to about 25 μM, or about 6 μM to about 25 μM. In some embodiments,the invention provides methods for increasing the activity and/orstability of an ALDC enzyme comprising adding a metal ion at aconcentration of about 25 μM to about 150 μM in a media. In someembodiments, the invention provides methods for increasing the activityand/or stability of an ALDC enzyme comprising adding a metal ion at aconcentration of about 100 μM to about 20 mM. In some embodiments, theinvention provides methods for increasing the activity and/or stabilityof an ALDC enzyme comprising adding a metal ion at a concentration ofabout 1 mM to about 5 mM. In some embodiments, the metal ion is selectedfrom the group consisting of Zn²⁺, Mg²⁺, Mn²⁺, Co²⁺, Cu²⁺, Ba²⁺, Ca²⁺and Fe²⁺ and combinations thereof. In some embodiments, the metal ion isselected from the group consisting of Zn²⁺, Cu²⁺, and Fe²⁺. In someembodiments, the metal ion is selected from the group consisting ofZn²⁺, Mn²⁺, and Co²⁺. In some embodiments, the metal ion is Zn²⁺ orMn²⁺. In some embodiments, the metal ion is Zn²⁺.

In some embodiments, the invention provides methods for increasing theactivity and/or stability of an ALDC enzyme in a composition comprisingALDC wherein said method comprises the step of adding a zinc to thecomposition so that said zinc is present in said composition at aconcentration of about 1 μM to about 200 mM, such as about 1 μM to about500 μM, or about 1 μM to about 300 μM, or about 6 μM to about 300 μM, orabout 1 μM to about 100 μM, or about 1 μM to about 50 μM, or about 10 μMto about 150 mM, or about 20 μM to about 120 mM, or about 25 μM to about100 mM, or about 25 μM to about 50 mM, or about 25 μM to about 20 mM, orabout 25 μM to about 50 μM, or about 100 μM to about 20 mM, or about 250μM to about 20 mM, or about 500 μM to about 20 mM, or about 1 mM toabout 20 mM, or about 1 mM to about 10 mM, or about 1 mM to about 5 mM,or about 5 mM to about 20 mM, or about 5 mM to about 10 mM. In someembodiments, the invention provides methods for increasing the activityand/or stability of an ALDC enzyme in a cultivation media comprising anALDC producing host cell wherein said method comprises the step ofadding a zinc at a concentration of about 1 μM to about 1 mM, such asabout 1 μM to about 300 μM, about 6 μM to about 300 μM, about 25 μM toabout 150 μM, or about 60 μM to about 150 μM. In some embodiments, theinvention provides methods for increasing the activity and/or stabilityof an ALDC enzyme in a fermentation and/or maturation media comprisingan ALDC enzyme wherein said method comprises the step of adding a zincto the media so that said zinc is present in said media at aconcentration of about 1 μM to about 300 μM, such as about 6 μM to about300 μM, about 1 μM to about 100 μM, about 1 μM to about 50 μM, about 1μM to about 25 μM, or about 6 μM to about 25 μM. In some embodiments,methods for increasing the activity and/or stability of an ALDC enzymecomprise adding a zinc to a media so that the zinc is at a concentrationof about 25 μM to about 150 μM in the media. In some embodiments,methods for increasing the activity and/or stability of an ALDC enzymecomprise adding a zinc at a concentration of about 100 μM to about 20mM. In some embodiments, methods for increasing the activity and/orstability of an ALDC enzyme comprise adding a zinc at a concentration ofabout 1 mM to about 5 mM. In some embodiments, methods for increasingthe activity and/or stability of an ALDC enzyme comprise adding zinc ata molar ratio of zinc to ALDC enzyme that is higher than 1 such as 2:1,or 3:1, or 5:1, or 10:1, or 20:1 or 30:1, or 50:1, or 60:1, or 100:1, or150:1, or 200:1 or 250:1 in said composition. In some embodiments,methods for increasing the activity and/or stability of an ALDC enzymecomprise adding zinc at a molar ratio of zinc to ALDC enzyme of 5:1 orhigher in said composition. In some embodiments, methods for increasingthe activity and/or stability of an ALDC enzyme comprise adding zinc ata molar ratio of zinc to ALDC enzyme of 10:1 or higher in saidcomposition. In some embodiments, methods for increasing the activityand/or stability of an ALDC enzyme comprise adding zinc at a molar ratioof zinc to ALDC enzyme of 20:1 or higher in said composition. In someembodiments, methods for increasing the activity and/or stability of anALDC enzyme comprise adding zinc at a molar ratio of zinc to ALDC enzymeof 30:1 or higher in said composition.

In some embodiments, the metal ion is added (e.g. as a supplement) to acultivation media during the production of said ALDC enzyme by an ALDCproducing host cell. In some embodiments, the metal ion is added at aconcentration of about 0.1 μM to about 1 mM, such as about 25 μM toabout 150 μM, or about 40 μM to about 150 μM, or about 60 μM to about150 μM, or about 25 μM to about 50 μM, or 30 μM to about 40 μM. In someembodiments, the metal ion is selected from the group consisting ofZn²⁺, Mg²⁺, Mn²⁺, Co²⁺, Cu²⁺, Ba²⁺, Ca²⁺ and Fe²⁺ and combinationsthereof. In some embodiments, the metal ion is selected from the groupconsisting of Zn²⁺, Cu²⁺, and Fe²⁺. In some embodiments, the metal ionis selected from the group consisting of Zn²⁺, Mn²⁺, and Co²⁺. In someembodiments, the metal ion is Zn²⁺ or Mn²⁺. In some embodiments, themetal ion is Zn²⁺. Thus, in some embodiments zinc is added (e.g. as asupplement) to a cultivation media during the production of said ALDCenzyme by an ALDC producing host cell at a concentration of 1 μM toabout 1 mM, such as 25 μM to about 150 μM, or about 40 μM to about 150μM, or 60 μM to about 150 μM.

In some embodiments, the host cell is a Bacillus host cell. In someembodiments, Bacillus host cell is Bacillus subtilis.

In some embodiments, the metal ion is added in the fermentation mediaduring production of a fermented beverage. In some embodiments, themetal ion is added in the fermentation media during beer and/or wineand/or cider and/or perry and/or sake fermentation. In some embodiments,the metal ion is selected from the group consisting of Zn²⁺, Mg²⁺, Mn²⁺,CO²⁺, Cu²⁺, Ba²⁺, Ca²⁺ and Fe²⁺ and combinations thereof. In someembodiments, the metal ion is selected from the group consisting ofZn²⁺, Cu²⁺, and Fe²⁺. In some embodiments, the metal ion is selectedfrom the group consisting of Zn²⁺, Mn²⁺, and Co²⁺. In some embodiments,the metal ion is Zn²⁺ or Mn²⁺. In some embodiments, the metal ion isZn²⁺. Thus, in some embodiments, zinc is added in a fermentation mediaduring beer and/or wine and/or cider and/or perry and/or sakefermentation. In some embodiments, zinc is added at a concentration ofabout 1 μM to about 1 mM, such as about 1 μM to about 300 μM, or about 6μM to about 300 μM, or about 1 μM to about 100 μM, or 25 μM to about 50μM, or 30 μM to about 40 μM, or 1 μM to about 50 μM, or 6 μM to about 50μM, or 1 μM to about 25 μM, or 6 μM to about 25 μM. In some embodimentszinc and the ALDC enzyme are added in a composition, wherein zinc ispresent in said composition at a concentration of 0.1 μM to about 200 mMor 1 μM to about 200 mM, or 0.1 mM to about 120 mM, such as 1 mM toabout 20 mM, or 1 mM to about 10 mM, or 1 mM to 5 mM. In someembodiments zinc and the ALDC enzyme are added in a composition, whereinthe molar ratio of zinc to ALDC enzyme in the composition is higher than1 such as 2:1, or 3:1, or 5:1, or 10:1, or 20:1 or 30:1, or 50:1, or60:1.

In some embodiments, the metal ion is added in the maturation mediaduring production of a fermented beverage. In some embodiments, themetal ion is added the maturation media during beer and/or wine and/orcider and/or perry and/or sake fermentation. In some embodiments, themetal ion is selected from the group consisting of Zn²⁺, Mg²⁺, Mn²⁺,Co²⁺, Cu²⁺, Ba²⁺, Ca²⁺ and Fe²⁺ and combinations thereof. In someembodiments, the metal ion is selected from the group consisting ofZn²⁺, Cu²⁺, and Fe²⁺. In some embodiments, the metal ion is selectedfrom the group consisting of Zn²⁺, Mn²⁺, and Co²⁺. In some embodiments,the metal ion is Zn²⁺ or Mn²⁺. In some embodiments, the metal ion isZn²⁺. Thus, in some embodiments, zinc is added in a maturation mediaduring beer and/or wine and/or cider and/or perry and/or sakefermentation. In some embodiments, zinc is added at a concentration of 1μM to about 1 mM, such as 1 μM to about 300 μM, or about 6 μM to about300 μM, or about 1 μM to about 100 μM, or 25 μM to about 50 μM, or 30 μMto about 40 μM, or 1 μM to about 50 μM, or 6 μM to about 50 μM, or 1 μMto about 25 μM, or 6 μM to about 25 μM. In some embodiments zinc andALDC are added in a composition, wherein zinc is present in saidcomposition at a concentration of 0.1 μM to about 200 mM, or 1 μM toabout 200 mM, or 0.25 mM to about 120 mM, such as 1 mM to about 20 mM,or 1 mM to about 10 mM, or 1 mM to about 5 mM. In some embodiments zincand the ALDC enzyme are added in a composition, wherein the molar ratioof zinc to ALDC enzyme in the composition is higher than 1 such as 2:1,or 3:1, or 5:1, or 10:1, or 20:1 or 30:1, or 50:1, or 60:1.

In some embodiments, a method of producing acetoin is provided in thedisclosure. In some embodiments, a method of decomposing acetolactate isprovided in the disclosure. In some embodiments, acetolactate isdecomposed to acetoin. The methods involve the step of treating asubstrate with an ALDC enzyme and a metal ion, wherein the metal ion ispresent at a concentration of about 1 μM to about 200 mM, such as about1 μM to about 500 μM, or about 1 μM to about 300 μM, or about 6 μM toabout 300 μM, or about 1 μM to about 100 μM, or about 1 μM to about 50μM, or 6 μM to about 50 μM, or 6 μM to about 25 μM, or about 10 μM toabout 150 mM, or about 20 μM to about 120 mM, or about 25 μM to about100 mM, or about 25 μM to about 50 mM, or about 25 μM to about 20 mM, orabout 25 μM to about 50 μM, or about 100 μM to about 20 mM, or about 250μM to about 20 mM, or about 1 mM to about 20 mM, or about 1 mM to about5 mM. In some embodiments the metal ion and the ALDC enzyme are added ina composition, where the metal ion is present in said composition at aconcentration of 0.1 μM to about 200 mM, or 1 μM to about 200 mM, or0.25 mM to about 120 mM, such as 1 mM to about 20 mM, or 1 mM to about 5mM. In some embodiments the metal ion and the ALDC enzyme are added in acomposition, wherein the molar ratio of metal ion to ALDC enzyme in thecomposition is higher than 1 such as 2:1, or 3:1, or 5:1, or 10:1, or20:1 or 30:1, or 50:1, or 60:1. In some embodiments, the metal ion isselected from the group consisting of Zn²⁺, Mg²⁺, Mn²⁺, Co²⁺, Cu²⁺,Ba²⁺, Ca²⁺ and Fe²⁺ and combinations thereof. In some embodiments, themetal ion is selected from the group consisting of Zn²⁺, Cu²⁺, and Fe²⁺.In some embodiments, the metal ion is selected from the group consistingof Zn²⁺, Mn²⁺, and Co²⁺. In some embodiments, the metal ion is Zn²⁺ orMn²⁺. In some embodiments, the metal ion is Zn²⁺. Thus, in someembodiments, the methods involve the step of treating a substrate withan ALDC enzyme and zinc, wherein said zinc is present at a concentrationof about 1 μM to about 1 mM, such as 1 μM to about 300 μM, or about 6 μMto about 300 μM, or 1 μM to about 100 μM, or 6 μM to about 100 μM, or 6μM to about 50 μM, or 6 μM to about 25 μM. In some embodiments zinc andthe ALDC enzyme are added in a composition, where zinc is present insaid composition at a concentration of 0.1 μM to about 200 mM, or 1 μMto about 200 mM, or 0.25 mM to about 120 mM, such as 1 mM to about 20mM, or 1 mM to about 5 mM. In some embodiments zinc and the ALDC enzymeare added in a composition, wherein the molar ratio of zinc to ALDCenzyme in the composition is higher than 1 such as 2:1, or 3:1, or 5:1,or 10:1, or 20:1 or 30:1, or 50:1, or 60:1.

In some embodiments a method of producing acetoin during the productionof a fermented beverage is provided in the disclosure. In someembodiments, a method of decomposing acetolactate during the productionof a fermented beverage is provided in the disclosure. In someembodiments, acetolactate is decomposed to acetoin.

Fermented Products

In one aspect the present invention relates to a process for producingfermented alcoholic products with a low diacetyl content by fermentationof a carbohydrate containing substrate with a microorganism. As usedherein, a fermented alcoholic product with “low diacetyl content” refersto a fermented alcoholic product (e.g. a beer and/or a wine and/or acider and/or a perry and/or sake) produced by fermentation of acarbohydrate containing substrate with a composition comprising ALDC inthe presence of a metal ion (such as zinc) wherein the diacetyl levelsare lower when compared to the fermented alcoholic produced byfermentation of a carbohydrate containing substrate with a compositioncomprising ALDC in the absence of a metal ion (such as zinc) under thesame fermentation conditions (e.g. same temperature and for the samelength of time). Examples of fermented alcoholic products with lowdiacetyl content are fermented alcoholic products in which the levels ofdiacetyl are less than about 1 ppm and/or the diacetyl levels are belowabout 0.5 mg/L. In one embodiment, the diacetyl levels are less thanabout 0.5 ppm, or less than about 0.1 ppm, or less than about 0.05 ppm,or less than about 0.01 ppm, or less than about 0.001 ppm. In oneembodiment, the diacetyl levels are about less than 0.1 mg/L, or aboutless than 0.05 mg/L, or about less than 0.01 mg/L or about less than0.001 mg/L.

When carbohydrate containing substrates, such as wort (e.g. worts withlow malt content) or fruit juices (such as grape juice, apple juice orpear juice), are fermented with yeast or other microorganisms, variousprocesses take place in addition to the alcohol fermentation which maycause generation of undesired by-products, e.g., the formation ofdiacetyl which has a strong and unpleasant smell even in very lowconcentrations. Alcoholic beverages, such as beer or wine or cider orperry or sake, may thus have an unacceptable aroma and flavor if thecontent of diacetyl considerably exceeds certain limits, e.g., in thecase of some beers about 0.1 ppm.

Formation of diacetyl is also disadvantageous in the industrialproduction of ethanol because it is difficult to separate diacetyl fromethanol by distillation. A particular problem arises in the preparationof absolute ethanol where ethanol is dehydrated by azeotropicdistillation with benzene. Diacetyl will accumulate in the benzene phaseduring the azeotropic distillation which may give rise to mixtures ofdiacetyl and benzene which makes it difficult to recover the benzeneused for the azeotropic distillation.

The conventional brewing of beer comprises fermenting the wort with asuitable species of yeast, such as Saccharomyces cerevisae orSaccharomyces carlsbergensis.

Typically in conventional brewing, the fermentation is usually effectedin two steps, a main fermentation of a duration of normally 5 to 12 daysand a secondary fermentation—a so-called maturation process-which maytake from up to 12 weeks. During the main fermentation most of thecarbohydrates in the wort are converted to ethanol and carbon dioxide.Maturation is usually effected at a low temperature in the presence of asmall residual amount of yeast. The purposes of the maturation are,inter alia, to precipitate undesirable, high molecular weight compoundsand to convert undesirable compounds to compounds, such as diols, whichdo not affect flavor and aroma. For example butanediol, the finalproduct of the conversion of α-acetolactate and diacetyl in beer, istypically reported as a compound with neutral sensory characteristics.The term “fermentation media” as used herein refers to a mediumcomprising carbohydrate containing substrates which can be fermented byyeast or other microorganisms to produce, for example, beer or wine orcider or perry or sake. Examples of fermentation media include: wort,and fruit juices (such as grape juice, apple juice and pear juice).Example 9 details an example of suitable wort. The term “maturationmedia” as used herein refers to a medium comprising carbohydratecontaining substrates which have been fermented by yeast or othermicroorganisms to produce, for example, beer or wine or cider or perryor sake. Examples of maturation media include partially fermented wortand fruit juices (such as grape juice, apple juice and pear juice). Insome embodiments, the invention provides a fermentation or maturationmedia for an ALDC producing host cell comprising a metal ion at aconcentration of about 1 μM to about 300 μM. In some embodiments, theinvention provides a fermentation or maturation media for an ALDCproducing host cell comprising a metal ion at a concentration of about 6μM to about 25 μM.

In some aspects, the present invention relates to the use of acomposition as described herein in beer and/or wine and/or cider and/orperry and/or sake fermentation. In some embodiments, the presentinvention comprises the use of the ALDC compositions described herein todecompose acetolactate during beer and/or wine and/or cider and/or perryand/or sake fermentation or maturation. Also, the invention comprisesthe use of ALDC derivative according to the invention to decomposeacetolactate during beer and/or wine and/or cider and/or perry and/orsake fermentation or maturation.

In some embodiments, the methods of the invention are thus characterizedby the treatment of a substrate with a composition comprising ALDC or anALDC derivative as described herein during or in continuation of afermentation process, e.g., maturation.

Thus, in some embodiments, acetolactate is enzymatically decarboxylatedto acetoin, the result being that when undesirable, the formation ofdiacetyl from acetolactate is avoided. In some embodiments, otherenzymes are used in combination with ALDC for the conversion ofα-acetolactate. Examples of such enzymes include, but are not limitedto, acetolactate reductoisomerases or isomerases.

In some embodiments, the ALDC and/or ALDC derivative compositionsdescribed herein are used together with ordinary yeast in batchfermentation.

Instead of using the enzyme in a free state, it may be used in animmobilized state, the immobilized enzyme being added to the wort duringor in continuation of the fermentation (e.g., during maturation). Theimmobilized enzyme may also be maintained in a column through which thefermenting wort or the beer is passed. The enzyme may be immobilizedseparately, or coimmobilized yeast cells and acetolactate decarboxylasemay be used.

In some embodiments, the ALDC and/or ALDC derivative compositionsdescribed herein are used during beer and/or wine and/or cider and/orperry and/or sake fermentation or maturation to reduce the diacetyllevels to below about 1 ppm, or about less than 0.5 ppm, or about lessthan 0.1 ppm, or about less than 0.05 ppm or about less than 0.01 ppm,or about less than 0.001 ppm. In some embodiments, the ALDC and/or ALDCderivative compositions described herein are used during beer and/orwine and/or cider and/or perry and/or sake fermentation or maturation toreduce the diacetyl levels to below 0.1 ppm.

In some embodiments, the ALDC and/or ALDC derivative compositionsdescribed herein are used during beer and/or wine and/or cider and/orperry and/or sake fermentation or maturation to reduce VDK content below0.1 mg/L, or about less than 0.05 mg/L, or less than 0.01 mg/L or lessthan 0.001 mg/L. Total VDK refers to the amount of Diacetyl plus2,3-pentanedione. In some embodiments, the ALDC and/or ALDC derivativecompositions described herein are used during beer and/or wine and/orcider and/or perry and/or sake fermentation or maturation to reduceTotal VDK content below 0.1 mg/L.

In some embodiments, the ALDC and/or ALDC derivative compositionsdescribed herein are used during beer and/or wine and/or cider and/orperry and/or sake fermentation or maturation to reduce the diacetyllevels to below about 0.5 mg/L, or about less than 0.1 mg/L, or aboutless than 0.05 mg/L, or about less than 0.01 mg/L or about less than0.001 mg/L. In some embodiments, the ALDC and/or ALDC derivativecompositions described herein are used during beer and/or wine and/orcider and/or perry and/or sake fermentation or maturation to reduce thediacetyl levels to below 0.1 mg/L.

In some embodiments, the ALDC and/or ALDC derivative compositionsdescribed herein are used during beer and/or wine and/or cider and/orperry and/or sake fermentation or maturation to reduce other vicinaldiketones (such as 2,3 pentanedione) to below 0.1 mg/L, or about lessthan 0.05 mg/L, or less than 0.01 mg/L or less than 0.001 mg/L.

The processes of the invention can not only be used in connection withthe brewing of beer, but is also suitable for the production of anysuitable alcoholic beverage where a reduction in diacetyl levels orother vicinal diketones is desirable (e.g. wine, sake, cider, perry,etc.). In some embodiments, the processes of the invention can be usedin the production of wine where similar advantages are obtained, inparticular a reduction in the maturation period and a simplification ofthe process. Of special interest in this context is the use ofacetolactate converting enzymes in connection with the so-calledmalo-lactic fermentation. This process which is affected bymicroorganisms as species of Leuconostoc, Lactobacillus or Pediococcusis carried out after the main fermentation of wine in order to increasethe pH of the product as well as its biological stability and to developthe flavor of the wine. Moreover, it is highly desirable to carry outthe fermentation since it makes possible rapid bottling and therebyimproves the cash-flow of wineries substantially. Unfortunately,however, the process may give rise to off-flavors due to diacetyl, theformation of which can be reduced with the aid of acetolactateconverting enzymes.

Thus, in some embodiments, the processes of the invention provide forthe production of alcoholic beverages with lower content of diacetyl,wherein the time required for producing the alcoholic beverages withlower content of diacetyl is reduced by at least 10%, or at least 20% orat least 30%, or at least 40%, or at least 50%, or at least 60%, or atleast 70%, or at least 80%, or at least 90% when compared to a processwithout the use of the ALDC and/or ALDC derivative compositionsdescribed herein. In some embodiments, the processes of the inventionprovide for the production of alcoholic beverages with lower content ofdiacetyl when compared to a process without the use of the ALDC and/orALDC derivative compositions described herein, wherein a maturation stepis completely eliminated.

In some embodiments, the ALDC and/or ALDC derivative compositionsdescribed herein are used during a fermentation process (e.g. beerand/or wine and/or cider and/or perry and/or sake fermentation), suchthat the time required for the fermentation process is reduced by atleast 10%, or at least 20% or at least 30%, or at least 40%, or at least50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%,when compared to a process without the use of the ALDC and/or ALDCderivative compositions described herein. In some embodiments, theprocesses of the invention provide for the production of alcoholicbeverages with lower content of diacetyl when compared to a processwithout the use of the ALDC and/or ALDC derivative compositionsdescribed herein, wherein a maturation step is completely eliminated.

In some embodiments, the ALDC and/or ALDC derivative compositionsdescribed herein are used during a maturation or conditioning process(e.g. beer maturation/conditioning), such that the time required for thematuration or conditioning process is reduced by at least 10%, or atleast 20% or at least 30%, or at least 40%, or at least 50%, or at least60%, or at least 70%, or at least 80%, or at least 90%, when compared toa process without the use of the ALDC and/or ALDC derivativecompositions described herein. In some embodiments, the processes of theinvention provide for the production of alcoholic beverages with lowercontent of diacetyl when compared to a process without the use of theALDC and/or ALDC derivative compositions described herein, wherein amaturation step is completely eliminated.

Further, in some embodiments, the processes described herein can be usedto advantage for industrial preparation of ethanol as fermentationproducts are obtained without or practically without any content ofdiacetyl, which simplifies the distillation process, especially in caseof azeotropic for the preparation of absolute ethanol, i.e. pureanhydrous ethanol.

In some embodiments, the invention provides methods for beer and/or wineand/or cider and/or perry and/or sake production comprising adding acomposition comprising an ALDC enzyme and metal ion to a media (such asa fermentation and/or a maturation media) for the beer and/or wineand/or cider and/or perry and/or sake so that the metal ion is presentin said media at a concentration of about 0.1 μM to about 500 μM, orabout 0.1 μM to about 300 μM, or about 0.1 μM to about 50 μM, or about 1μM to about 500 μM, or about 1 μM to about 300 μM, or about 6 μM toabout 300 μM, or about 1 μM to about 100 μM, or about 1 μM to about 50μM, or about 6 μM to about 50 μM, or about 6 μM to about 25 μM, or about10 μM to about 150 mM, or about 20 μM to about 120 mM, or about 25 μM toabout 100 mM, or about 25 μM to about 50 mM, or about 25 μM to about 20mM, or about 25 μM to about 50 μM, or about 100 μM to about 20 mM, orabout 250 μM to about 20 mM, or about 1 mM to about 20 mM, or about 1 mMto about 5 mM. In some embodiments, the metal ion is selected from thegroup consisting of Zn²⁺, Mg²⁺, Mn²⁺, Co²⁺, Cu²⁺, Ba²⁺, Ca²⁺ and Fe²⁺and combinations thereof. In some embodiments, the metal ion is selectedfrom the group consisting of Zn²⁺, Cu²⁺, and Fe²⁺. In some embodiments,the metal ion is selected from the group consisting of Zn²⁺, Mn²⁺, andCo²⁺. In some embodiments, the metal ion is Zn²⁺ or Mn²⁺. In someembodiments, the metal ion is Zn²⁺. In some embodiments, the inventionprovides methods for beer and/or wine and/or cider and/or perry and/orsake production comprising adding a composition comprising an ALDCenzyme and zinc to a media (such as a fermentation and/or a maturationmedia) for the beer and/or wine and/or cider and/or perry and/or sakewherein said zinc is present in the composition at a concentration ofabout 0.25 mM to about 200 mM, such as about 1 mM to about 20 mM, orabout 1 mM to about 5 mM. In some embodiments, the invention providesmethods for beer and/or wine and/or cider and/or perry and/or sakeproduction comprising adding a composition comprising an ALDC enzyme andzinc to a media (such as a fermentation and/or a maturation media) forthe beer and/or wine and/or cider and/or perry and/or sake, wherein themolar ratio of zinc to ALDC enzyme that is higher than 1 such as 2:1, or3:1, or 5:1, or 10:1, or 20:1 or 30:1, or 50:1, or 60:1. In someembodiments, the ALDC enzyme and said zinc are added during afermentation process and/or a maturation process. In some embodiments,the ALDC enzyme is added at a concentration of about 0.01 g to about 10g, or about 0.5 g to about 10 g, or about 1 g to about 5 g perhectoliter of beer and/or wine and/or cider and/or perry and/or sakeferment. As used herein the term “beer and/or wine and/or cider and/orperry and/or sake ferment” may be interchangeable with the term media.In some embodiments, the activity of said ALDC enzyme is in the range of1000 to 2500 Units per mg of protein.

In some embodiments, the invention provides methods for beer and/or wineand/or cider and/or perry and/or sake production comprising adding acomposition comprising an ALDC enzyme and metal ion to a media (such asa fermentation and/or a maturation media) for the beer and/or wineand/or cider and/or perry and/or sake, wherein the metal ion is presentin said composition at a concentration of about 1 μM to about 200 mM, orabout 100 μM to about 200 mM, and the composition comprising the ALDCenzyme and the metal ion are added at a concentration of about 0.01 g toabout 10 g per hectoliter of beer and/or wine and/or cider and/or perryand/or sake ferment. In some embodiments, the invention provides methodsfor beer and/or wine and/or cider and/or perry and/or sake productioncomprising adding a composition comprising ALDC enzyme and metal ion toa media (such as a fermentation and/or a maturation media) for the beerand/or wine and/or cider and/or perry and/or sake, wherein the metal ionis present in said composition at a concentration of about 1 μM to about200 mM, or about 100 μM to about 200 mM, and the composition comprisingthe ALDC enzyme and the metal ion are added at a concentration of about0.5 g to about 10 g per hectoliter of beer and/or wine and/or ciderand/or perry and/or sake ferment. In some embodiments, the inventionprovides methods for beer and/or wine and/or cider and/or perry and/orsake production comprising adding a composition comprising an ALDCenzyme and metal ion to a media (such as a fermentation and/or amaturation media) for the beer and/or wine and/or cider and/or perryand/or sake, wherein the metal ion is present in said composition at aconcentration of about 1 μM to about 200 mM or about 100 μM to about 200mM, and the composition comprising the ALDC enzyme and the metal ion areadded at a concentration of about 1 g to about 5 g per hectoliter ofbeer and/or wine and/or cider and/or perry and/or sake ferment. In someembodiments, the invention provides methods for beer and/or wine and/orcider and/or perry and/or sake production comprising adding acomposition comprising an ALDC enzyme and metal ion to a media (such asa fermentation and/or a maturation media) for the beer and/or wineand/or cider and/or perry and/or sake, wherein the metal ion is presentin said composition at a concentration of about 1 μM to about 200 mM, orabout 100 μM to about 200 mM, and the composition comprising the ALDCenzyme and the metal ion are added at a concentration of about 1 g toabout 2 g per hectoliter of beer and/or wine and/or cider and/or perryand/or sake ferment. In some embodiments the metal ion is present in thecomposition at a concentration of about 1 mM to about 20 mM, or about 1mM to about 10 mM, or about 1 mM to about 5 mM. In some embodiments, themetal ion is selected from the group consisting of Zn²⁺, Mg²⁺, Mn²⁺,Co²⁺, Cu²⁺, Ba²⁺, Ca²⁺ and Fe²⁺ and combinations thereof. In someembodiments, the metal ion is selected from the group consisting ofZn²⁺, Cu²⁺, and Fe²⁺. In some embodiments, the metal ion is selectedfrom the group consisting of Zn²⁺, Mn²⁺, and Co²⁺. In some embodiments,the metal ion is Zn²⁺ or Mn²⁺. In some embodiments, the metal ion isZn²⁺. In some embodiments, the activity of said ALDC enzyme is in therange of 950 to 2500 Units per mg of protein or 1000 to 2500 Units permg of protein or 1500 to 2500 Units per mg of protein.

In some embodiments, the invention provides methods for beer and/or wineand/or cider and/or perry and/or sake production comprising adding anALDC enzyme and metal ion in a composition to a media (such as afermentation and/or a maturation media) for the beer and/or wine and/orcider and/or perry and/or sake, wherein the molar ratio of the metal ionto the ALDC enzyme is higher than 1, and the composition comprising theALDC enzyme and the metal ion are added at a concentration of about 0.01g to about 10 g per hectoliter of beer and/or wine and/or cider and/orperry and/or sake ferment. In some embodiments, the invention providesmethods for beer and/or wine and/or cider and/or perry and/or sakeproduction comprising adding an ALDC enzyme and metal ion in acomposition to a media (such as a fermentation and/or a maturationmedia) for the beer and/or wine and/or cider and/or perry and/or sake,wherein the molar ratio of the metal ion to the ALDC enzyme is higherthan 1, and the composition comprising the ALDC enzyme and the metal ionare added at a concentration of about 0.5 g to about 10 g per hectoliterof beer and/or wine and/or cider and/or perry and/or sake ferment. Insome embodiments, the invention provides methods for beer and/or wineand/or cider and/or perry and/or sake production comprising adding anALDC enzyme and metal ion in a composition to a media (such as afermentation and/or a maturation media) for the beer and/or wine and/orcider and/or perry and/or sake, wherein the molar ratio of the metal ionto the ALDC enzyme is higher than 1, and the composition comprising theALDC enzyme and the metal ion are added at a concentration of about 1 gto about 5 g per hectoliter of beer and/or wine and/or cider and/orperry and/or sake ferment. In some embodiments, the invention providesmethods for beer and/or wine and/or cider and/or perry and/or sakeproduction comprising adding an ALDC enzyme and metal ion in acomposition to a media (such as a fermentation and/or a maturationmedia) for the beer and/or wine and/or cider and/or perry and/or sake,wherein the molar ratio of the metal ion to the ALDC enzyme is higherthan 1, and the composition comprising the ALDC enzyme and the metal ionare added at a concentration of about 1 g to about 2 g per hectoliter ofbeer and/or wine and/or cider and/or perry and/or sake ferment. In someembodiments, the molar ratio of the metal ion to the ALDC enzyme is 2:1,or 3:1, or 5:1, or 10:1, or 20:1 or 30:1, or 50:1, or 60:1, or higher.In some embodiments, the metal ion is selected from the group consistingof Zn²⁺, Mg²⁺, Mn²⁺, Co²⁺, Cu²⁺, Ba²⁺, Ca²⁺ and Fe²⁺ and combinationsthereof. In some embodiments, the metal ion is selected from the groupconsisting of Zn²⁺, Cu²⁺, and Fe²⁺. In some embodiments, the metal ionis selected from the group consisting of Zn²⁺, Mn²⁺, and Co²⁺. In someembodiments, the metal ion is Zn²⁺ or Mn²⁺. In some embodiments, themetal ion is Zn²⁺. In some embodiments, the activity of said ALDC enzymeis in the range of 950 to 2500 Units per mg of protein or 1000 to 2500Units per mg of protein or 1500 to 2500 Units per mg of protein.

Production of ALDC Enzymes

In one aspect, the description relates to a nucleic acid capable ofencoding an ALDC enzyme as described herein. In a further aspect, thedescription relates to an expression vector or plasmid comprising such anucleic acid, or capable of expressing an ALDC enzyme as describedherein. In one aspect, the expression vector or plasmid comprises apromoter derived from Trichoderma such as a T. reesei cbh1-derivedpromoter. In a further aspect, the expression vector or plasmidcomprises a terminator derived from Trichoderma such as a T. reeseicbh1-derived terminator. In yet a further aspect, the expression vectoror plasmid comprises one or more selective markers such as Aspergillusnidulans amdS and pyrG. In another aspect, the expression vector orplasmid comprises one or more telomere regions allowing for anon-chromosomal plasmid maintenance in a host cell.

In one aspect, the description relates to a host cell havingheterologous expression of an ALDC enzyme as herein described. In afurther aspect, the host cell is a fungal cell. In yet a further aspect,the fungal cell is of the genus Trichoderma. In yet a further aspect,the fungal cell is of the species Trichoderma reesei or of the speciesHypocrea jecorina. In another aspect, the host cell comprises,preferably is transformed with, a plasmid or an expression vector asdescribed herein.

In some embodiments, the host cell is a bacterial host cell such asBacillus. In some embodiments the ALDC enzyme is produced by cultivationof a Bacillus subtilis strain containing a gene encoding and expressingan ALDC enzyme (e.g. ALDC of Bacillus brevis). Examples of such hostcells and cultivation thereof are described in DK149335B.

Examples of suitable expression and/or integration vectors are providedin Sambrook et al. (1989) supra, and Ausubel (1987) supra, and van denHondel et al. (1991) in Bennett and Lasure (Eds.) More GeneManipulations In Fungi, Academic Press pp. 396-428 and U.S. Pat. No.5,874,276. Reference is also made to the Fungal Genetics Stock CenterCatalogue of Strains (FGSC, http://www.fgsc.net) for a list of vectors.Particularly useful vectors include vectors obtained from for exampleInvitrogen and Promega. Suitable plasmids for use in bacterial cellsinclude pBR322 and pUC19 permitting replication in E. coli and pE194 forexample permitting replication in Bacillus. Other specific vectorssuitable for use in E. coli host cells include vectors such as pFB6,pBR322, pUC18, pUC100, pDONR™201, 10 pDONR™221, pENTR™, pGEM®3 Z andpGEM®4 Z.

Specific vectors suitable for use in fungal cells include pRAX, ageneral purpose expression vector useful in Aspergillus, pRAX with aglaA promoter, and in Hypocrea/Trichoderma includes pTrex3 g with a cbh1promoter.

In some embodiments, the host cells are fungal cells and optionallyfilamentous fungal host cells. The term “filamentous fungi” refers toall filamentous forms of the subdivision Eumycotina (see, Alexopoulos,C. J. (1962), Introductory Mycology, Wiley, New York). These fungi arecharacterized by a vegetative mycelium with a cell wall composed ofchitin, cellulose, and other complex polysaccharides. The filamentousfungi of the present disclosure are morphologically, physiologically,and genetically distinct from yeasts. Vegetative growth by filamentousfungi is by hyphal elongation and carbon catabolism is obligatoryaerobic. In the present disclosure, the filamentous fungal parent cellmay be a cell of a species of, but not limited to, Trichoderma (e.g.,Trichoderma reesei, the asexual morph of Hypocrea jecorina, previouslyclassified as T. longibrachiatum, Trichoderma viride, Trichodermakoningii, Trichoderma harzianum) (Sheir-Neirs et al., Appl. Microbiol.Biotechnol. 20:46-53 (1984); ATCC No. 56765 and ATCC No. 26921),Penicilliurn sp., Humicola sp. (e.g., H. insolens, H. lanuginosa and H.grisea), Chrysosporium sp. (e.g., C. lucknowense), Gliocladium sp.,Aspergillus sp. (e.g., A. oryzae, A. niger, A sojae, A. japonicus, A.nidulans, and A. awamori) (Ward et al., Appl. Microbiol. Biotechnol.39:738-743 (1993) and Goedegebuur et al., Curr. Genet. 41:89-98 (2002)),Fusarium sp., (e.g., F. roseum, F. graminum, F. cerealis, F. oxysporum,and F. venenatum), Neurospora sp., (N. crassa), Hypocrea sp., Mucor sp.(M miehei), Rhizopus sp., and Emericella sp. (see also, Innis et al.,Science 228:21-26 (1985)). The term “Trichoderma” or “Trichoderma sp.”or “Trichoderma spp.” refer to any fungal genus previously or currentlyclassified as Trichoderma.

In some embodiments, the host cells will be gram-positive bacterialcells. Non-limiting examples include strains of Streptomyces (e.g., S.lividans, S. coelicolor, and S. griseus) and Bacillus. As used herein,“the genus Bacillus” includes all species within the genus “Bacillus,”as known to those of skill in the art, including but not limited to B.subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus,B. alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B.megaterium, B. coagulans, B. circulans, B. lautus, and B. thuringiensis.It is recognized that the genus Bacillus continues to undergotaxonomical reorganization. Thus, it is intended that the genus includespecies that have been reclassified, including but not limited to suchorganisms as B. stearothermophilus, which is now named “Geobacillustearothermophilus.”

In some embodiments, the host cell is a gram-negative bacterial strain,such as E. coli or Pseudomonas sp. In other embodiments, the host cellsmay be yeast cells such as Saccharomyces sp., Schizosaccharomyces sp.,Pichia sp., or Candida sp. In other embodiments, the host cell will be agenetically engineered host cell wherein native genes have beeninactivated, for example by deletion in bacterial or fungal cells. Whereit is desired to obtain a fungal host cell having one or moreinactivated genes known methods may be used (e.g., methods disclosed inU.S. Pat. No. 5,246,853, U.S. Pat. No. 5,475,101, and WO 92/06209). Geneinactivation may be accomplished by complete or partial deletion, byinsertional inactivation or by any other means that renders a genenonfunctional for its intended purpose (such that the gene is preventedfrom expression of a functional protein). In some embodiments, when thehost cell is a Trichoderma cell and particularly a T. reesei host cell,the cbh1, cbh2, egl1 and egl2 genes will be inactivated and/or deleted.Exemplary Trichoderma reesei host cells having quad-deleted proteins areset forth and described in U.S. Pat. No. 5,847,276 and WO 05/001036. Inother embodiments, the host cell is a protease deficient or proteaseminus strain. The term “protease deficient” or a “protease minus strain”as used herein refers to a host cell derived or derivable from aparental cell wherein the host cell comprises one or more geneticalterations that causes the host cells to produce a decreased amount ofone or more proteases (e.g. functional proteases) when compared to theparental cell; preferably said host cell is deficient in one or moreproteases selected from the group consisting of WprA, Vpr, Epr, IspA,Bpr, NprE, AprE, ampS, aprX, bpf, clpCP, clpEP, clpXP, codWX, lonA,lonB, nprB, map, mlpA, mpr, pepT, pepF, dppA, yqyE, tepA, yfiT, yflG,ymfF, ypwA, yrrN, yrrO, and ywaD. a variant host cell derived from aparental cell is provided, the variant host cell comprises one or moregenetic alterations that causes cells of the variant strain to produce adecreased amount of one or more proteases when compared to the parentalcell.

Introduction of a DNA construct or vector into a host cell includestechniques such as transformation; electroporation; nuclearmicroinjection; transduction; transfection, (e.g., lipofection-mediatedand DEAE-Dextrin mediated transfection); incubation with calciumphosphate DNA precipitate; high velocity bombardment with DNA-coatedmicroprojectiles; and protoplast fusion. General transformationtechniques are known in the art (see, e.g., Ausubel et al. (1987) supra,chapter 9; and Sambrook et al. (1989) supra, and Campbell et al., Curr.Genet. 16:53-56 (1989)).

Transformation methods for Bacillus are disclosed in numerous referencesincluding Anagnostopoulos C. and J. Spizizen, J. Bacteriol. 81:741-746(1961) and WO 02/14490. Transformation methods for Aspergillus aredescribed in Yelton et al., Proc. Natl. Acad. Sci. USA 81:1470-1474(1984); Berka et al., (1991) in Applications of Enzyme Biotechnology,Eds. Kelly and Baldwin, Plenum Press (NY); Cao et al., Protein Sci.9:991-1001 (2000); Campbell et al., Curr. Genet. 16:53-56 (1989), and EP238 023. The expression of heterologous protein in Trichoderma isdescribed in U.S. Pat. No. 6,022,725; U.S. Pat. No. 6,268,328; Harkki etal. Enzyme Microb. Technol. 13:227-233 (1991); Harkki et al.,BioTechnol. 7:596-603 (1989); EP 244,234; EP 215,594; and Nevalainen etal., “The Molecular Biology of Trichoderma and its Application to theExpression of Both Homologous and Heterologous Genes”, in MolecularIndustrial Mycology, Eds. Leong and Berka, Marcel Dekker Inc., NY (1992)pp. 129-148). Reference is also made to WO96/00787 and Bajar et al.,Proc. Natl. Acad. Sci. USA 88:8202-8212 (1991) for transformation ofFusarium strains.

In one aspect, the description relates to a method of isolating an ALDCenzyme as defined herein, the method comprising the steps of inducingsynthesis of the ALDC enzyme in a host cell as defined herein havingheterologous expression of said ALDC enzyme and recovering extracellularprotein secreted by said host cell, and optionally purifying the ALDCenzyme. In a further aspect, the description relates to a method forproducing an ALDC enzyme as defined herein, the method comprising thesteps of inducing synthesis of the ALDC enzyme in a host cell as definedherein having heterologous expression of said ALDC enzyme, andoptionally purifying the ALDC enzyme. In a further aspect, thedescription relates to a method of expressing an ALDC enzyme as definedherein, the method comprising obtaining a host cell as defined herein,or any suitable host cells as known by a person of ordinary skill in theart, and expressing the ALDC enzyme from said host cell, and optionallypurifying the ALDC enzyme. In another aspect, the ALDC enzyme as definedherein is the dominant secreted protein. In some embodiments, metal ionssuch as Zn²⁺, Mg²⁺, Mn²⁺, Co²⁺, Cu²⁺, Ba²⁺, Ca²⁺ and Fe²⁺ andcombinations thereof are added to the media (such as a cultivationand/or a fermentation and/or a maturation media) during and/or afterALDC production to increase the recovered yields from microorganisms.

In some embodiments, the invention provides a cultivation media for anALDC producing host cell comprising a metal ion at a concentration ofabout 1 μM to about 1 mM. In some embodiments, the invention provides acultivation media for an ALDC producing host cell comprising a metal ionat a concentration of about 25 μM to about 150 μM. In some embodiments,the invention provides a cultivation media for an ALDC producing hostcell comprising a metal ion at a concentration of about 25 μM to about50 μM. In some embodiments, the invention provides a cultivation mediafor an ALDC producing host cell comprising a metal ion at aconcentration of about 30 μM to about 40 μM. In some embodiments, theinvention provides a cultivation media for an ALDC producing host cellcomprising a metal ion at a concentration of about 40 μM to about 150μM. In some embodiments, the invention provides a cultivation media foran ALDC producing host cell comprising a metal ion at a concentration ofabout 60 μM to about 150 μM. In some embodiments, the metal ion isselected from the group consisting of Zn²⁺, Mg²⁺, Mn²⁺, Co²⁺, Cu²⁺, andFe²⁺ and combinations thereof. In some embodiments, the metal ion isselected from the group consisting of Zn²⁺, Cu²⁺, and Fe²⁺. In someembodiments, the metal ion is selected from the group consisting ofZn²⁺, Mn²⁺, and Co²⁺. In some embodiments, the metal ion is Zn²⁺or Mn²⁺.In some embodiments, the metal ion is Zn²⁺. In some embodiments, theactivity of said ALDC enzyme is in the range of 950 to 2500 Units per mgof protein or 1000 to 2500 Units per mg of protein or 1500 to 2500 Unitsper mg of protein. The term “ALDC producing host cell” as used hereinrefers to a host cell capable of expressing ALDC enzyme when said hostcell is cultured under conditions permitting the expression of thenucleic acid sequence encoding ALDC. The nucleic acid sequence encodingALDC enzyme may be heterologous or homologous to the host cell. In someembodiments, the ALDC producing host cell is Bacillus subtilis. In someembodiments, the ALDC producing host cell is Bacillus subtiliscomprising a gene encoding and expressing ALDC enzyme wherein the ALDCenzyme comprises an amino acid sequence having at least 70%, 75%, 80%,85%, 90%, 95%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 2, SEQ IDNO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, or any functionalfragment thereof. In some embodiments, the ALDC producing host cell isBacillus subtilis comprising a nucleic acid sequence encoding ALDCwherein said nucleic acid sequence encoding ALDC has at least 70%, 75%,80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 1,SEQ ID NO: 4, SEQ ID NO: 6 or any functional fragment thereof. In someembodiments, the ALDC producing host cell is Bacillus subtiliscomprising a gene encoding ALDC derived from Bacillus brevis.

EXAMPLES

The present disclosure is described in further detail in the followingexamples, which are not in any way intended to limit the scope of thedisclosure as claimed. The attached figures are meant to be consideredas integral parts of the specification and description of thedisclosure. The following examples are offered to illustrate, but not tolimit the claimed disclosure.

Example 1—Heterologous Expression of Acetolactate Decarboxylase, aldB

The Brevibacillus brevis (which may be referred to as Bacillus brevis)acetolactate decarboxylases (ALDC) aldB gene was previously identified(Diderichsen et al., J Bacteriol. (1990) 172(8): 4315), with thesequence set forth as UNIPROT Accession No. P23616.1. The sequence ofthis gene, aldB, is depicted in SEQ ID NO:1. The nucleotides highlightedin bold and underlined are the nucleotides which encode the signalpeptide.

sets forth the nucleotide sequence of the aldB gene: SEQ ID NO: 1atgaaaaaaaatatcatcacttctatcacatctctggctctggttgccgg gctgtctttgactgcttttgcagctacaacggctactgtaccagcaccacctgccaagcaggaatccaaacctgcggttgccgctaatccggcaccaaaaaatgtactgtttcaatactcaacgatcaatgcactcatgcttggacagtttgaaggggacttgactttgaaagacctgaagctgcgaggcgatatggggcttggtaccatcaatgatctcgatggagagatgattcagatgggtacaaaattctaccagatcgacagcaccggaaaattatcggagctgccagaaagtgtgaaaactccatttgcggttactacacatttcgagccgaaagaaaaaactacattaaccaatgtgcaagattacaatcaattaacaaaaatgcttgaggagaaatttgaaaacaagaacgtcttttatgccgtaaagctgaccggtacctttaagatggtaaaggctagaacagttccaaaacaaaccagaccttatccgcagctgactgaagtaaccaaaaaacaatccgagtttgaatttaaaaatgttaagggaaccctgattggcttctatacgccaaattatgcagcagccctgaatgttcccggattccatctccacttcatcacagaggataaaacaagtggcggacacgtattaaatctgcaatttgacaacgcgaatctggaaatttctccgatccatgagtttgatgtacaattgccgcacacagatgattttgcccactctgatctgacacaagttactactagccaagtacaccaagctgagtcagaaa gaaaataasets forth an example of a nucleotide sequence encoding an ace tolactate decarboxylase-the nucleotides highlighted in bold and underlined arethe nucleotides whichencode the signal peptide: SEQ ID NO: 6atgaaaaaaaatatcatcacttctatcacatctctggctctcgttgccgggctgctttgactgcttttgcagctacaacggctactgtaccagcaccacctgccaagcaggaatccaaacctgtggttgccgctaatccggcaccaaaaaatgtactgtttcaatactcaacgatcaatgcactcatgcttggacagtttgaaggggacttgactttgaaagacctgaagctacgaggcgatatggggct tggtaccatcaatgatctcgatggagagatgattcagatgggtacaaaattctaccagatcgacagcaccggaaaattatccgagctgccagaaagtgtgaaaactccatttgcggttactacacatttcgagccgaaagaaaaaactacattaaccaatgtgcaagattacaatcaattaacaaaaatgcttgaggagaaatttgaaaacaagaacgtcttttatgccgtaaagctgaccggtacctttaagatggtaaaggctagaacagttccaaaacaaaccagaccttatccgcagctgactgaagtaaccaaaaaacaatccgagtttgaatttaaaaatgttaagggaaccctgattggcttctatacgccaaattatgcagcagccctgaatgttcccggattccatctccacttcatcacagaggataaaacaagtggcggacacgtattaaatctgcaatttgacaacgcgaatctggaaatttctccgatccatgagtttgatgtacaattgccgcacacagatgattttgcccactctgatctgacacaagttactactagccaagtacaccaagctgagtcagaaag aaaataa

The proenzyme encoded by the aldB gene is depicted in SEQ ID NO: 2. Atthe N-terminus, the protein has a signal peptide with a length of 24amino acids as predicted by SignalP-NN (Emanuelsson et al., NatureProtocols (2007) 2: 953-971). This signal peptide sequence is underlinedand is in bold in SEQ ID NO:2. The presence of a signal peptideindicates that this acetolactate decarboxylase, aldB is a secretedenzyme. The sequence of the predicted, fully processed mature chain(aldB, 261 amino acids) is depicted in SEQ ID NO: 3.

sets forth the amino acid sequence of the aceto-lactate decarboxylase (ALDC) precursor aldB: SEQ ID NO: 2MKKNIITSITSLALVAGLSLTAFA ATTATVPAPPAKQESKPAVAANPAPKNVLFQYSTINALMLGQFEGDLTLKDLKLRGDMGLGTINDLDGEMIQMGTKFYQIDSTGKLSELPESVKTPFAVTTHFEPKEKTTLTNVQDYNQLTKMLEEKFENKNVFYAVKLTGTFKMVKARTVPKQTRPYPQLTEVTKKQSEFEFKNVKGTLIGFYTPNYAAALNVPGFHLHFITEDKTSGGHVLNLQFDNANLEISPIHEFDVQLPHTDDFAHSDLTQVTTSQVHQAESERKsets forth an example of an amino acid sequence ofthe acetolactate decarboxylase (ALDC) precursoraldB-the signal peptide sequence is underlined and is in bold:SEQ ID NO: 7 MKKNIITSITSLALVAGLSLTAFA ATTATVPAPPAKQESKPVVAANPAPKNVLFQYSTINALMLGQFEGDLTLKDLKLRGDMGLGTINDLDGEMIQMGTKFYQIDSTGKLSELPESVKTPFAVTTHFEPKEKTTLTNVQDYNQLTKMLEEKFENKNVFYAVKLTGTFKMVKARTVPKQTRPYPQLTEVTKKQSEFEFKNVKGTLIGFYTPNYAAALNVPGFHLHFITEDKTSGGHVLNLQFDNANLEISPIHEFDVQLPHTDDFAHSDLTQVTTSQVHQAESERKsets forth the predicted amino acid sequence ofthe mature acetolactate decarboxylase (ALDC) aldB (261 amino acids):SEQ ID NO: 3 ATTATVPAPPAKQESKPAVAANPAPKNVLFQYSTINALMLGQFEGDLTLKDLKLRGDMGLGTINDLDGEMIQMGTKFYQIDSTGKLSELPESVKTPFAVTTHFEPKEKTTLTNVQDYNQLTKMLEEKFENKNVFYAVKLTGTFKMVKARTVPKQTRPYPQLTEVTKKQSEFEFKNVKGTLIGFYTPNYAAALNVPGFHLHFITEDKTSGGHVLNLQFDNANLEISPIHEFDVQLPHTDDFAHSDLTQVTT SQVHQAESERKsets forth an example of the predicted amino acidsequence of the mature acetolactate decarboxylase(ALDO aldB (261 amino acids): SEQ ID NO: 8ATTATVPAPPAKQESKPVVAANPAPKNVLFQYSTINALMLGQFEGDLTLKDLKLRGDMGLGTINDLDGEMIQMGTKFYQIDSTGKLSELPESVKTPFAVTTHFEPKEKTTLTNVQDYNQLTKMLEEKFENKNVFYAVKLTGTFKMVKARTVPKQTRPYPQLTEVTKKQSEFEFKNVKGTLIGFYTPNYAAALNVPGFHLHFITEDKTSGGHVLNLQFDNANLEISPIHEFDVQLPHTDDFAHSDLTQVTT SQVHQAESERK

The aldB gene from the strain Brevibacillus brevis encoding anacetolactate decarboxylases enzyme (ALDC) was produced in B. subtilisusing an integrated expression cassette consisting of the B. subtilisaprE promoter, the B. subtilis aprE signal peptide sequence, the maturealdB and a BPN′ terminator. This cassette was cloned in the head to headorientation with respect to the expression cassette and the aldBexpression cassette introduced into B. subtilis by homologousrecombination.

A map of the vector containing the aldB gene (RIHI-aldB) is shown inFIG. 1. The nucleotide mature sequence of the aldB gene in plasmidRIHI-aldB is depicted in SEQ ID NO:4.

gctacaacggctactgtaccagcaccacctgccaagcaggaatccaaacctgcggttgccgctaatccggcaccaaaaaatgtactgtttcaatactcaacgatcaatgcactcatgcttggacagtttgaaggggacttgactttgaaagacctgaagctgcgaggcgatatggggcttggtaccatcaatgatctcgatggagagatgattcagatgggtacaaaattctaccagatcgacagcaccggaaaattatcggagctgccagaaagtgtgaaaactccatttgeggttactacacatttcgagccgaaagaaaaaactacattaaccaatgtgcaagattacaatcaattaacaaaaatgcttgaggagaaatttgaaaacaagaacgtcttttatgccgtaaagctgaccggtacttttaagatggtaaaggctagaacagttccaaaacaaaccagaccttatccgcagctgactgaagtaaccaaaaaacaatccgagtttgaatttaaaaatgttaagggaaccctgattggcttctatacgccaaattatgcagcagccctgaatgttcccggattccatctccacttcatcacagaggataaaacaagtggcggacacgtattaaatctgcaatttgacaacgcgaatctggaaatttctccgatccatgagtttgatgttcaattgccgcacacagatgattttgcccactctgatctgacacaagttactactagccaagtacaccaagctgagtcagaaagaaaa

The amino acid sequence of the aldB precursor protein expressed fromplasmid RIHI-aldB is depicted in SEQ ID NO:5.

ATTATVPAPPAKQESKPAV AANPAPKNVLFQYSTINALMLGQFEGDLTLKDLKLRGDMGLGTINDLDGEMIQMGTKFYQIDSTGKLSELPESVKTPFAVTTHFEPKEKTTLTNVQDYNQLTKMLEEKFENKNVFYAVKLTGTFKMVKARTVPKQTRPYPQLTEVTKKQSEFEFKNVKGTLIGFYTPNYAAALNVPGFHLHFITEDKTSGGHVLNLQFDNANLEISPIHEFDVQLPHTDDFAHSDLTQVTTSQVHQAESERKThe signal peptide sequence is in bold lowercase italics in SEQ ID NO:5.

Small Scale Culture Conditions

To produce aldB, a B. subtilis strain transformant containing aldBexpression cassette was cultured in 15 mL Falcon tubes for 5 hours inTSB (broth) with 300 ppm beta-chloro-D-alanine (CDA), and 300 μL of thispre-culture was added to a 500 mL flask filled with 30 mL of cultivationmedia (described below) supplemented with 300 ppm CDA and 50 μM Zn²⁺.The flasks were incubated for 24, 48 and 72 hours at 33° C. withconstant rotational mixing at 180 rpm. Cultures were harvested bycentrifugation at 14500 rpm for 20 minutes in conical tubes. The culturesupernatants were used for protein determination and assays. Thecultivation media was an enriched semi-defined media based on MOPsbuffer, with urea as major nitrogen source, maltrin as the main carbonsource.

Fed-Batch Fermentation Conditions

To produce aldB, a B. subtilis strain transformant containing aldBexpression cassette was cultured in a 250 mL flasks containing 30 mL ofcomplex medium with 300 ppm beta-chloro-D-alanine (CDA). The flask wasincubated for 6 hours at 37° C. with constant rotational mixing at 180rpm.

The culture was transferred to a stirred fermentor containing 7 litersof sterilized media components as described in table 1 below.Temperature was controlled to 37° C.; pH was controlled to 7,5 usingammonium hydroxide as alkaline titrant; dissolved oxygen was maintainedat 40% or higher by maintaining an airflow of 7 liter/min, a constantoverpressure of 1 bar and adjusting stirring rate. When initial glucosewas exhausted a feeding profile feeding a 60% glucose solution into thefermentor was initiated (initial feeding rate was 20 g/h linearlyincreasing to 32.8 g/h over 7 hours and kept constant at that rate untilfermentation termination).

Total fermentation time was 44 hours.

TABLE 1 Media recipe for ALDC fermentation Recipe Conc (g/kg Componentbroth) Soy Meal 50.0 Citric acid 0.10 Magnesium sulfate heptahydrate2.29 Potassium Phosphate, Mono Basic 5.44 Ferrous sulfate, heptahydrate0.029 Manganese Sulfate Mono hydrate 0.051 Zinc sulphate heptahydrate0.001 Glucose mono hydrate 1.10 Anti foam agent 3.00

Example 2—Protein Determination Methods Protein Determination by StainFree Imager Criterion

Protein was quantified by SDS-PAGE gel and densitometry using Gel Doc™EZ imaging system. Reagents used in the assay: Concentrated (2×) LaemmliSample Buffer (Bio-Rad, Catalogue #161-0737); 26-well XT 4-12% Bis-TrisGel (Bio-Rad, Catalogue #345-0125); protein markers “Precision PlusProtein Standards” (Bio-Rad, Catalogue #161-0363); protein standard BSA(Thermo Scientific, Catalogue #23208) and SimplyBlue Safestain(Invitrogen, Catalogue #LC 6060. The assay was carried out as follow: Ina 96well-PCR plate 504, diluted enzyme sample were mixed with 50 μLsample buffer containing 2.7 mg DTT. The plate was sealed by Microseal‘B’ Film from Bio-Rad and was placed into PCR machine to be heated to70° C. for 10 minutes. After that the chamber was filled by runningbuffer, gel cassette was set. Then 10 μL of each sample and standard(0.125-1.00 mg/mL BSA) was loaded on the gel and 5 μL of the markerswere loaded. After that the electrophoresis was run at 200 V for 45 min.Following electrophoresis the gel was rinsed 3 times 5 min in water,then stained in Safestain overnight and finally destained in water. Thenthe gel was transferred to Imager. Image Lab software was used forcalculation of intensity of each band. By knowing the protein amount ofthe standard sample, the calibration curve can be made. The amount ofsample can be determined by the band intensity and calibration curve.The protein quantification method was employed to prepare samples ofaldB acetolactate decarboxylases enzyme used for assays shown insubsequent Examples.

Example 3—Activity Assay Method Spectrophotometric Assay ofα-Acetolactate Decarboxylase

α-Acetolactate decarboxylase (ALDC) catalyses the decarboxylation ofα-acetolactate to acetoin. The reaction product acetoin can bequantified colourimetrically. Acetoin mixed with α-naphtol and creatineforms a characteristic red color absorbing at OD522 nm. ALDC activitywas calculated based on OD_(522 nm) and an acetoin calibration curve.The assay was carried out as follows: 20 mM acetolactate substrate wasprepared by mixing 100 μL ethyl-2-acetoxy-2-methylacetoacetate (Sigma,Catalogue#220396) with 3.6 mL 0.5 M NaOH at 10° C. for 10 min. 20 mL 50mM MES pH 6.0 was added, pH was adjusted to pH 6.0 and volume adjustedto 25 mL with 50 mM MES pH 6.0. 80 μL 20 mM acetolactate substrate wasmixed with 20 μL enzyme sample diluted in 50 mM MES, pH 6.0, 0.6 M NaCl,0.05% BRIJ 35 and 0.01% BSA. The substrate/enzyme mixture was incubatedat 30° C. for 10 min. Then 16 μL substrate/enzyme mixture wastransferred to 200 μL 1 M NaOH, 1.0% α-naphtol (Sigma, Catalogue#33420)and 0.1% creatine (Sigma, Catalogue# C3630). The substrate/enzyme/colorreagent mixture was incubated at 30° C. for 20 min and then OD_(522 nm)was read. One unit of ALDC activity is defined as the amount of enzymewhich produces 1 μmole acetoin per minute under the conditions of theassay.

Example 4—Zinc Influence on Activity of ALDC The Presence of Zn²⁺ DuringShake Flask Fermentation

ZnSO₄ was added to the cultivation media for small scale fermentationsas described in Example 1 at concentrations ranging from 0 mM to 800 μM.aldB. B. subtilis transformant containing aldB expression cassette wascultured in 15 mL Falcon tubes for 5 hours in TSB (broth) with 300 ppmbeta-chloro-D-alanine (CDA), and 300 μL of this pre-culture was added toa each 500 mL flask filled with 30 mL of cultivation media supplementedwith 300 ppm CDA and containing 0, 5, 25, 50, 100, 200, 400 and 800 μMZn²⁺. The flasks were incubated for 24 and 48 hours at 33° C. withconstant rotational mixing at 180 rpm. Cultures were harvested bycentrifugation at 14500 rpm for 20 minutes in conical tubes. The culturesupernatants were used for protein determination and ALDC activityassays (Table 2 and 3). It is clearly seen that increasing theconcentration of Zn²⁺ in the fermentation media increased the total ALDCactivity and specific activity of expressed aldB enzyme.

TABLE 2 ALDC activity of aldB fermentation samples with varying levelsof Zn²⁺ after 24 and 48 hours of fermentation ALDC activity ZnSO₄ in 24hours of 48 hours of sample fermentation fermentation μM U/mL U/mL aldB0 298.6 235.3 5 371.9 354.8 25 533.9 808.0 50 606.4 965.3 100 633.4668.8 200 617.6 629.5 400 691.1 488.5 800 428.1 474.0

TABLE 3 ALDC activity, aldB protein and specific activity of aldBfermentation samples with varying levels of Zn²⁺ after 48 hours offermentation ZnSO₄ in sample ALDC activity AldB protein Specificactivity μM U/mL mg/mL U/mg aldB 0 235.3 0.626 375.9 5 354.8 0.736 482.125 808.0 1.437 562.3 50 965.3 1.573 609.9 100 668.8 0.957 698.9

Example 5—Modulation of the Specific Activity of aldB Samples Zinc DoseResponse of ALDC at 50° C.

ZnSO₄ was added to ALDC fermentation sample to produce concentrationsranging from 0 mM to 20 mM. The samples were kept at 50° C. for 60 minand then activity was measured as described in Example 3. Relative to asample with no zinc added samples with >0.5 mM zinc had activity >200%higher. FIG. 2 shows results.

Example 6—Modulation of the Specific Activity of aldB Ferment

AldB activity was produced as described in (Example 1) and activitydetermined as described in (Example 3). An AldB ferment sample wasdiluted in 50 mM MES pH 6.0, 0.6 M NaCl, 0.05% Brij, 0.01% BSA (bovineserum albumin) to proximately 1000 U/mL and then incubated with varyingconcentration of ZnSO₄ in the following steps: 0, 0.25, 0.5, 1, 2, 5,7.5, 10, 20, 40, 60, 80, 100, 120 mM. All samples were incubated at 4°C. for 68 hours, then centrifuged and supernatant analyzed for activityand protein by Criterion SDS-PAGE analysis. The results of the SDS-PAGEanalysis is shown in FIG. 3 and the determined activity, proteinconcentration and specific activity are given in table 4. It can be seenthat the ALDC activity increases with increasing concentration of ZnSO₄in the sample until approximately 5-10 mM from where it is more or lessconstant with the higher concentrations of ZnSO₄. The concentration ofaldB protein in the sample was determined to be more or less constant inall samples indicating an apparent increase in the specific activityfrom 968 U/mg in the sample without ZnSO₄ up to 2200-2400 U/mg insamples with 5 mM ZnSO₄ or more after incubation.

TABLE 4 ALDC activity, aldB protein concentration and specific activityof aldB sample incubated with ZnSO₄ at 4° C. for 68 hours. ZnSO₄ in ALDCAldB Specific sample activity protein activity mM U/mL mg/mL U/mg aldB 0556 0.57 968 0.25 875 0.63 1387 0.5 949 0.63 1512 1 1058 0.59 1792 21271 0.60 2124 5 1411 0.60 2354 7.5 1485 0.64 2311 10 1520 0.64 2368 201492 0.64 2344 40 1499 0.67 2239 60 1495 0.64 2345 80 1500 0.65 2324 1001607 0.64 2520 120 1520 0.69 2209

Example 7—Purification of aldB

Production of aldB in B. subtilis was carried out as described inexample (1). Fermentation media was clarified by centrifugation (4000rpm at 15 min.) and filtration (VacuCap 90, 0.2 μm). The sample wasdesalted on PD10 column, prepared as described by the manufacturer andequilibrated with 20 mM Tris/HCl, pH 8.0. The eluate was kept on iceafterwards before further purified by anion exchange chromatography on aSource 15Q XK26/15. The column was equipped on an AKTA explorer systemused for protein purification and according to the method described bythe manufacturer (GE healthcare, USA—Cat. No. 18-1112-41).

The column was prepared as described by the manufacturer andequilibrated with 20 mM Tris/HCl, pH 8.0 (buffer A). The desalted samplewas applied to the column at a flow rate of 5 mL/min and the column waswashed with buffer A. The bound proteins were eluted with a lineargradient of 0-0.30 M NaCl in 20 mM Tris/HCl, pH 8.0 (7 mL/min, 40 min).During the entire run, fractions of approx. 10-20 mL were collected.

Fractions from purification were analysed by SDS-PAGE using XcellMini-cell and according to the method described by the manufacturer(Invitrogen, USA—Cat. No. EI0001) using: Nu-PAGE gel (Invitrogen,USA—Cat. No. NP0321), MES running buffer (Invitrogen, USA—Cat. No.NP0002), See Blue standard marker (Invitrogen, USA—Cat. No. LC5925) andStained with Coomasie Brilliant-Blue.

AldB was observed to bind at the Source 15Q column and was eluted withapproximately 30 mM NaCl. AldB fractions were collected, concentrated(on a 10 kDa UF Vivaspin) and desalted in 25 mM HEPES buffer containing150 mM NaCl, pH 8.0. The estimated purity of aldB was above 95% and theresult of SDS-page analysis of the purified aldB is shown in FIG. 4.

Example 8—Modulation of the Specific Activity of Purified aldB

The purified aldB protein was stripped of divalent ions by incubation ofaldB (approximately 2 mg/mL) with 20 mM EDTA in 0.2× assay buffer (50 mMIVIES pH 6.0, 0.6 M NaCl, 0.05% Brij, 0.01% BSA) at 37° C. overnight.The EDTA treated material was desalted on a PD10 column prepared asdescribed by the manufacturer and equilibrated with 50 mM IVIES pH 6.0,0.6 M NaCl, 0.05% Brij, 0.01% BSA. ALDC activity and the concentrationof AldB protein in the sample before and after EDTA treatment and thedesalting column were determined as described in example (2 and 3) andthe results are shown in table 5.

TABLE 5 ALDC activity, aldB protein concentration and specific activityof purified aldB before and after the EDTA-desalting procedure. ALDCAldB protein Specific Activity concentration Activity U/mL mg/mL U/mgAldB purified 5187 5.6 928 AldB purified - EDTA treated and 9 1.0 9desalted

50 μL of the EDTA treated and desalted aldB sample was mixed with 4504,of 0.2× assay buffer (50 mM MES pH 6.0, 0.6 M NaCl, 0.05% Brij, 0.01%BSA) with varying ZnSO₄ in the step: 0, 0.05, 0.1, 0.5, 1, 2, 5, 10 and20 mM respectively. Samples were incubated at 37° C. overnight. Thesamples were centrifuged at 13000 rpm for 10 min and ALDC activity andthe concentration of AldB protein were determined in the supernatant.The results are shown in table 6. It can be seen that the ALDC activityincreases with increasing concentration of ZnSO₄ during incubation. Theconcentration of aldB protein in the sample was determined to beconstant in all samples indicating an increase in the specific activityfrom 2 U/mg of aldB in the sample without ZnSO₄ up to 1800 U/mg aldB insamples with 5 mM ZnSO₄ or more after incubation.

TABLE 6 ALDC activity, aldB protein concentration and specific activityof purified aldB before and after incubation with ZnSO₄. Concentrationof Final ZnSO₄ in 0.2X concentration ALDC aldB protein Specific assaybuffer of ZnSO₄ Activity concentration activity mM mM U/mL mg/mL U/mg 00 0.1 0.092 2 0.05 0.045 4.4 0.092 48 0.1 0.09 26.1 0.092 283 0.5 0.45147.1 0.092 1599 1 0.9 157.7 0.092 1714 2 1.8 157.9 0.092 1716 5 4.5166.4 0.092 1809 10 9 174.6 0.092 1898 20 18 173.5 0.092 1886

In addition, 204, of the EDTA treated and desalted aldB sample was mixedwith 1804, of 50 mM MES pH 6.0 supplemented with either 10 mM ZnSO₄,MnSO₄ or CoCl₂ respectively. Samples were incubated at 37° C. andassayed after 45 minutes, 1 day, 2 days and 3 days. Results are shown intable 7. The activity increased for incubation with all three divalentions in the order Zn²⁺, Mn²⁺ and Co²⁺ compared to the control (additionof H₂O). After 45 min the activity was shown to decrease over time. Theaddition of Zn²⁺, Mn²⁺ and Co²⁺ all resulted in an increased stability(activity) after 3 days compared to the control with addition of H₂O.The residual activity measured after 3 days relative to the start were76.9%, 73.5% and 70.7% upon addition of Zn²⁺, Mn²⁺ and Co²⁺ compared to56.0% obtained with H₂O, as shown in table 7.

TABLE 7 ALDC activity after incubation at pH 6.0 with MnSO₄, CoCl₂ orZnSO₄ at various times. Activity U/mL H₂O Mn²⁺ Co²⁺ Zn²⁺ 45 min 21.8217.3 101.8 319.7  1 day 18.9 219.6 90.5 288.5  2 days 22.5 216.2 95.2295.4  3 days 12.2 153.6 74.8 245.7

Example 9—Reduction in Diacetyl and 2,3-Pentanedione During BeerFermentation by Use of aldB with Different Specific ALDC Activity

The objective of this analysis was to test different formulationvariants of aldB (acetolactate decarboxylase) ability to reducedevelopment of diacetyl and 2,3-pentanedione (Vicinal di-ketones, VDK)during a 7 days fermentation at 14° C.

Pure Malt Brew Analysis

1100 g Munton's Light Malt Extract (Batch XB 35189, expiry date 24 May2014) extract was dissolved in 3000 mL warm tapwater (45° C.). Thisslurry was stirred for about 10 min until the liquid was homogeneous andthe pH was adjusted to 5.2 with 2.5 M sulphuric acid. To the slurry wasadded 10 pellets of Bitter hops from Hopfenveredlung, St. Johann: Alphacontent of 16.0% (EBC 7.7 0 specific HPLC analysis, 1 Oct. 2013), thensplit in 500 mL blue-cap bottles and boiled for 1 hour to ensure proteinprecipitation and avoid potential microbial contamination. The filteredmalt extract (wort) was sampled for specific gravity and Free AminoNitrogen (FAN) determination. The final wort had an initial SpecificGravity of 1048 (i.e. 12° Plato). 200 g of the filtered wort were addedto a 500 mL conical flask (Fermenting Vessel; FV), and then cooled to13° C. Each conical flask was dosed with 0.5% W34/70 (Weihenstephan)freshly produced yeast (1.0 g yeast per 200 g wort). The enzymes weredosed on similar ALDC activity (0.03 U/mL wort, 8 ALDC Units per 200 gwort). The control fermentation vessel with no enzyme received an amountof deionized water corresponding to the amount of enzyme sample.

The wort samples were fermented in 500 mL conical flasks understandardised laboratory test conditions at 14° C. with gentle agitationat 150 rpm in an orbital incubator. When weight loss was less than 0.25g over 24 hours, fermentation temperature was decreased to 7° C.Fermentation was stopped after 7 days in total. 10 mL samples were takenout for diacetyl and 2,3-pentanedione analysis two times a day,preferably with 11 to 14 hours in between; at the end of fermentationonly 1 sample per day was taken. Yeast was allowed to settle beforetake-out and each sample was cooled at 10° C. for 10 minutes and thencentrifuged at 4000 rpm for 10 minutes at 8° C. to sediment any residualyeast. The supernatant was separated from the yeast sediment and samplesfor GC analysis were added 0.5 g NaCl per mL of sample. This slurry wastransferred to a headspace vial and heat-treated at 65° C. for 30minutes before analysis for diacetyl and 2,3-pentanedione were carriedout by gas chromatography with mass spectrometric detection (GCMS).

Analyses were carried out at an Agilent 6890N/5973N GC with CombiPALheadspace autosampler and MSChemStation acquisition and analysissoftware. The samples were equilibrated at 70° C. for 10 minutes before500 μl of the gas phase above the sample was injected onto a J&W122-0763 DB-1701column (60 m×0.25 mmID×1 μm). The injection temperaturewas 260° C. and the system was operated with a constant helium flow of 2mL/min. The oven temperature was: 50° C. (2 min), 160° C. (20° C./min),220° C. (40° C./min), hold 2 min. MS detection were made with 500 μL ata split ratio of 5:1 at selected ions. All sample were run in duplicatesand standards were made using tap water with the addition of diacetyl or2,3-pentanedione.

The concentration of a compound is calculated as

${{Compound}\mspace{14mu} \left( {{mg}\text{/}L} \right)} = {R\; F \times \frac{Area}{1000 \times W_{s}}}$

where,

RF is the response factor of acetic acid

Area is the GC-area of acetic acid

W_(s) is the amount of sample used (in mL)

The limit of diacetyl quantification was determined to 0.016 mg/L andthe limit of 2,3-pentanedione quantification was determined to 0.012mg/L.

To check that addition of ALDC enzymes did not influence the Real Degreeof Fermentation (RDF) and the produced alcohol by volume: RDF wasmeasured using an Anton Paar (DMA 5000) following Standard InstructionBrewing, 23.8580-B28 and alcohol by Standard Instruction Brewing,23.8580-B28.

Real degree of fermentation (RDF) value may be calculated according tothe equation below:

${R\; D\; F\mspace{14mu} (\%)} = {\left( {1 - \frac{R\; E}{{^\circ}\mspace{14mu} P_{initial}}} \right) \times 100}$

Where: RE=real extract=(0.1808°P_(initial))+(0.8192×° P_(final)),V_(initial) is the specific gravity of the standardised worts beforefermentation and ° P_(final) is the specific gravity of the fermentedworts expressed in degree Plato.

In the present context, Real degree of fermentation (RDF) was determinedfrom the specific gravity and alcohol concentration.

Specific gravity and alcohol concentration was determined on thefermented samples using a Beer Alcolyzer Plus and a DMA 5000 Densitymeter (both from Anton Paar, Gratz, Austria). Based on thesemeasurements, the real degree of fermentation (RDF) value was calculatedaccording to the equation below:

${R\; D\; F\mspace{14mu} (\%)} = {\frac{{O\; E} - {E(r)}}{O\; E} \times 100}$

Where: E(r) is the real extract in degree Plato (° P) and OE is theoriginal extract in ° P.

The ability to reduce development of diacetyl and 2,3-pentanedione(Vicinal di-ketones, VDK) during a 7 days fermentation at 14° C. wasstudied by addition of aldB with different specific ALDC activity(obtained by varying the ZnSO₄ concentration in the sample) is given intable 8. The aldB variant constitute: A) a crude preparation of aldB, B)a crude preparation of aldB including 20 mM ZnSO₄ added to the clarifiedbroth and C) a crude preparation of aldB including 50 μM ZnSO₄ added tothe fermentation media and 20 mM ZnSO₄ into the clarified broth.

TABLE 8 ALDC activity, aldB protein concentration and specific activityof aldB sample A, B and C. ALDC aldB Specific ALDC activity proteinactivity U/mL mg/mL U/mg aldB variant A 426 0.463 919 aldB variant B 5110.463 1103 aldB variant C 719 0.463 1552

The enzymes variants were dosed on similar ALDC activity 0.03 U/mL wortresulting in a different concentration of aldB protein in the wort, asseen in table 9. The calculated aldB protein concentration in the wortwas 33.4, 28.0 and 19.9 μg/L wort with addition of variant A, B and Crespectively.

TABLE 9 ALDC activity and aldB protein in wort with addition of aldBvariant A, B and C. Volume AldB ALDC Amount sample pre- Activity proteinin activity for pre-dilution dilution in wort wort U/mL g mL U/mL μg/lAldB variant A 426 0.721 50 0.03 33.4 AldB variant B 511 0.604 50 0.0328.0 AldB variant C 719 0.430 50 0.03 19.9

The fermentations were conducted and VDK development analysed asdescribed above. Fermentations with enzyme were compared to a controlwithout any enzyme added. The development of VDK is shown in FIG. 5.

All three aldB variants reduced the VDK development during fermentationcompared to control. The data suggest that the three variants performedvery similar, however with less increase in VDK during the fermentationperiod from 20 to 40 hours for variant C compared to A and B, but thisis close to the relative standard deviation of the analysis (RSD of7.5%). The relative reduction in VDK after 40 hours of fermentation was63.1%, 58.8% and 63.2% for variant A, B and C respectively. In addition,the fermentation time required to reach threshold level of 0.1 mg/ml VDKor lower, was observed to be approximately 112 hours for all threevariants. Thus, the higher specific activity of variant C compared to Aor B may enable similar VDK reduction with less aldB protein.

Example 10—Reduction in Diacetyl and 2,3-Pentanedione by aldB DuringBeer Fermentation with Various Concentration of Zinc in the Wort

The objective of this analysis was to test reduction of diacetyl and2,3-pentanedione (Vicinal di-ketones, VDK) by aldB during a 4 daysfermentation at 14° C. in presence of various level of ZnSO₄ in thewort.

The brew analysis setup was as described in example 9. A stock solutionof ZnSO₄ was made in cooled wort (0.686 g in 1000 mL wort) and appliedto the wort samples (prepared as described in example 9) beforefermentation resulting in the following Zn²⁺ concentrations 0, 0.1, 0.5,0.75, 1, 2.5, 5.0 and 20.0 ppm respectively. ALDC enzyme and yeast weresubsequently added to the samples. An ALDC enzymes preparation weredosed similarly to all samples (0.04 U/mL wort, corresponding to 8 ALDCunits per 200 g of wort). Each conical fermentation flask was dosed with0.5% W34/70 (Weihenstephan) freshly produced yeast (1.0 g yeast per 200g of wort) and analyses were carried out as described in example 9.Fermentations with enzyme were compared to a control without any enzymeadded. The development of VDK is shown in FIG. 6.

All aldB treated samples provided less increase in the VDK developmentduring fermentation compared to control. Increasing the concentration ofZn²⁺ in the samples resulted in less and less increase in VDKgeneration. Especially increasing the concentration of Zn²⁺ to 0.5 ppmor more greatly affected the enzymatic activity to avoid generation ofVDK. Supplementing the wort with 1 ppm Zn²⁺ or more ensured a VDK levelbelow the flavor threshold of 0.1 mg/L throughout the whole fermentationperiod.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

1. A composition comprising an acetolactate decarboxylase (ALDC) enzyme and zinc, wherein said zinc is present at a concentration of about 1 μM to about 200 mM.
 2. The composition of claim 1, wherein the zinc is present at a concentration of about 10 μM to about 150 mM, or about 20 μM to about 120 mM, or about 25 μM to about 100 mM, or about 25 μM to about 50 mM, or about 25 μM to about 20 mM, or about 25 μM to about 50 μM, or about 100 μM to about 20 mM, or about 250 μM to about 20 mM, or about 500 μM to about 20 mM, or about 1 mM to about 20 mM, or about 1 mM to about 10 mM, or about 1 mM to about 5 mM.
 3. The composition of claim 2, wherein the zinc (i) is present at a concentration of about 100 μM to about 10 mM; or (ii) is present at a concentration of about 1 mM to about 5 mM.
 4. The composition of claim 3, wherein the molar ratio of zinc to ALDC enzyme is (i) higher than 1; or (ii) 2:1 or higher; or (iii) 10:1 or higher; or (iv) 20:1 or higher; or (v) 30:1 or higher; or (vi) 60:1 or higher.
 5. The composition of claim 4, wherein said ALDC enzyme is an ALDC derivative.
 6. The composition of claim 5, wherein said ALDC derivative is an ALDC enzyme treated with glutaraldehyde.
 7. The composition of claim 6, wherein said ALDC enzyme is treated with glutaraldehyde at a concentration corresponding to about 0.1 to about 5 g of glutaraldehyde per g of pure ALDC enzyme.
 8. The composition of claim 7, wherein the activity of said ALDC enzyme is in the range of 950 to 2500 Units per mg of protein.
 9. The composition of claim 8, wherein the activity of said ALDC enzyme is in the range of 1000 to 2500 Units per mg of protein.
 10. The composition of claim 9 further comprising at least one additional enzyme or enzyme derivative selected from the group consisting of acetolactate reductoisomerases, acetolactate isomerases, amylase, glucoamylase, hemicellulase, cellulase, glucanase, pullulanase, isoamylase, endo-glucanase and related beta-glucan hydrolytic accessory enzymes, xylanase, xylanase accessory enzymes (for example, arabinofuranosidase, ferulic acid esterase, and xylan acetyl esterase) and protease.
 11. The composition of claim 1, wherein the ALDC enzyme is from Lactobacillus casei, Brevibacterium acetylicum, Lactococcus lactis, Leuconostoc lactis, Enterobacter aerogenes, Bacillus subtilis, Bacillus brevis, Lactococcus lactis DX, or Bacillus licheniformis.
 12. The composition of claim 11, wherein the ALDC enzyme is from Bacillus brevis or Bacillus licheniformis.
 13. The composition of claim 12, wherein said ALDC enzyme has an amino acid sequence having at least 80% identity with any one selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 8 or any functional fragment thereof.
 14. Use of the composition according to claim 13 in beer and/or wine and/or cider and/or perry and/or sake fermentation.
 15. A method for increasing the activity and/or stability of an ALDC enzyme in a composition comprising ALDC wherein said method comprises the step of adding zinc to the composition so that said zinc is present in said composition at a concentration of about 1 μM to about 200 mM.
 16. The method of claim 15, wherein the zinc is present in said composition at a concentration of about 10 μM to about 150 mM, or about 20 μM to about 120 mM, or about 25 μM to about 100 mM, or about 25 μM to about 50 mM, or about 25 μM to about 20 mM, or about 25 μM to about 50 μM, or about 100 μM to about 20 mM, or about 250 μM to about 20 mM, or about 500 μM to about 20 mM, or about 1 mM to about 20 mM, or about 1 mM to about 10 mM, or about 1 mM to about 5 mM.
 17. The method of claim 16, wherein the zinc is present in said composition at a concentration of (i) about 100 μM to about 10 mM; or (ii) about 1 mM to about 5 mM.
 18. The method of claim 16, wherein the molar ratio of zinc to ALDC enzyme is (i) higher than 1; or (ii) 2:1 or higher; or (iii) 10:1 or higher; or (iv) 20:1 or higher; or (v) 30:1 or higher; or (vi) 60:1 or higher in said composition.
 19. A method for increasing the activity and/or stability of an ALDC enzyme in a cultivation media comprising an ALDC producing host cell wherein said method comprises the step of adding zinc to the cultivation media as a supplement during the production of said ALDC enzyme by the ALDC producing host cell.
 20. The method of claim 19, wherein said zinc is added at a concentration of 1 μM to about 1 mM.
 21. The method of claim 20, wherein said zinc is added at a concentration of 25 μM to about 150 μM, or 60 μM to about 150 μM.
 22. The method of claim 21, wherein said host cell is a Bacillus host cell.
 23. The method of claim 22, wherein said Bacillus host cell is Bacillus subtilis.
 24. A method for increasing the activity and/or stability of an ALDC enzyme in a fermentation media and/or maturation media comprising an ALDC producing host cell wherein said method comprises the step of adding zinc to the fermentation media and/or maturation media during a beer and/or wine and/or cider and/or perry and/or sake fermentation.
 25. The method of claim 24, wherein said zinc: (i) is added at a concentration of about 1 μM to about 300 μM, such as about 6 μM to about 300 μM; or (ii) is added at a concentration of about 1 μM to about 50 μM; or (iii) is added at a concentration of about 1 μM to about 25 μM, preferably about 6 μM to about 25 μM.
 26. The method of claim 24, wherein said zinc is added as a composition comprising ALDC and zinc, wherein said zinc is present in said composition at a concentration of 1 mM to about 5 mM.
 27. A cultivation media for an ALDC producing host cell comprising zinc at a concentration of about 1 μM to about 1 mM; preferably said cultivation media comprises an ALDC producing host cell.
 28. The cultivation media of claim 27, comprising zinc at concentration of about 25 μM to about 150 μM.
 29. The cultivation media of claim 27, wherein said zinc is added at a concentration of 60 μM to about 150 μM.
 30. A beer and/or wine and/or cider and/or perry and/or sake fermentation media and/or maturation media comprising a composition comprising an ALDC enzyme and zinc wherein said composition comprises zinc at a concentration of about 1 μM to about 200 mM.
 31. The beer and/or wine and/or cider and/or perry and/or sake fermentation media and/or maturation media of claim 30, wherein: (i) zinc is present in the composition at a concentration of about 1 μM to about 300 μM, such as about 6 μM to about 300 μM; or (ii) zinc is present in the composition at a concentration of about 1 μM to about 50 μM, such as about 6 μM to about 50 μM, or about 6 μM to about 25 μM; or (iii) the zinc and the ALDC enzyme are added in a composition, wherein zinc is present in the composition at a concentration of about 1 mM to about 20 mM, such as 1 mM to about 5 mM; or (iv) the zinc and the ALDC enzyme are added in a composition, where the molar ratio of zinc to ALDC enzyme in the composition is higher than 1; or 2:1 or higher; or 10:1 or higher; or 20:1 or higher; or 30:1 or higher; or 60:1 or higher.
 32. The beer and/or wine and/or cider and/or perry and/or sake fermentation media and/or maturation media of claim 31, wherein the activity of said ALDC enzyme is in the range of 1000 to 2500 Units per mg of protein.
 33. The beer and/or wine and/or cider and/or perry and/or sake fermentation media and/or maturation media of claim 32, further comprising at least one additional enzyme or enzyme derivative selected from the group consisting of acetolactate reductoisomerases, acetolactate isomerases, amylase, glucoamylase, hemicellulase, cellulase, glucanase, pullulanase, isoamylase, endo-glucanase and related beta-glucan hydrolytic accessory enzymes, xylanase, xylanase accessory enzymes (for example, arabinofuranosidase, ferulic acid esterase, and xylan acetyl esterase) and protease.
 34. A composition comprising an ALDC enzyme, wherein said ALDC enzyme is in the range of 1000 to 2500 Units per mg of protein.
 35. The composition of claim 34, wherein the ALDC enzyme is from Lactobacillus casei, Brevibacterium acetylicum, Lactococcus lactis, Leuconostoc lactis, Enterobacter aerogenes, Bacillus subtilis, Bacillus brevis, Lactococcus lactis DX, or Bacillus licheniformis.
 36. The composition of claim 34, wherein the ALDC enzyme is from Bacillus brevis or Bacillus licheniformis.
 37. The composition of claim 36, wherein said ALDC enzyme has an amino acid sequence having at least 80% identity with any one selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 8 or any functional fragment thereof.
 38. The composition of claim 37, wherein: (i) zinc is present at a concentration of about 1 μM to about 200 mM; or (ii) the molar ratio of zinc to ALDC enzyme in the composition is higher than 1; or 2:1 or higher; or 10:1 or higher; or 20:1 or higher; or 30:1 or higher; or 60:1 or higher.
 39. A method for beer and/or wine and/or cider and/or perry and/or sake production comprising adding an ALDC enzyme and adding zinc to a media (such as a fermentation and/or a maturation media) for the beer and/or wine and/or cider and/or perry and/or sake during said beer and/or wine and/or cider and/or perry and/or sake production, so that said zinc is present in said media at a concentration of about 1 μM to about 300 μM, such as about 6 μM to about 300 μM.
 40. The method of claim 39, wherein said zinc is present in said media at a concentration of about 0.1 μM to about 50 μM, such as about 1 μM to about 50 μM, or about 6 μM to about 50 μM, or about 6 μM to about 25 μM.
 41. A method for beer and/or wine and/or cider and/or perry and/or sake production comprising adding a composition comprising an ALDC enzyme and zinc to a media (such as a fermentation and/or a maturation media) for the beer and/or wine and/or cider and/or perry and/or sake during said beer and/or wine and/or cider and/or perry and/or sake production, wherein (i) zinc is present in the composition at a concentration of about 1 mM to about 5 mM; or (ii) the molar ratio of zinc to ALDC enzyme in the composition is higher than 1; or 2:1 or higher; or 10:1 or higher; or 20:1 or higher; or 30:1 or higher; or 60:1 or higher.
 42. The method of claim 41, wherein said ALDC enzyme and said zinc are added during a fermentation process or a maturation process.
 43. The method of claim 42, wherein said ALDC enzyme is added at a concentration of about 0.5 g to about 10 g per hectoliter of beer and/or wine and/or cider and/or perry and/or sake ferment.
 44. The method of claim 43, wherein said ALDC enzyme is added at a concentration of about 1 g to about 5 g per hectoliter of beer and/or wine and/or cider and/or perry and/or sake ferment.
 45. The method of claim 44, wherein the activity of said ALDC enzyme is in the range of 1000 to 2500 Units per mg of protein.
 46. The method of claim 45, wherein the ALDC enzyme is from Lactobacillus casei, Brevibacterium acetylicum, Lactococcus lactis, Leuconostoc lactis, Enterobacter aerogenes, Bacillus subtilis, Bacillus brevis, Lactococcus lactis DX, or Bacillus licheniformis.
 47. The method of claim 45, wherein the ALDC enzyme is from Bacillus brevis or Bacillus licheniformis.
 48. The method of claim 45, wherein said ALDC enzyme has an amino acid sequence having at least 80% identity with any one selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 8 or any functional fragment thereof.
 49. A method for increasing the activity and/or stability of an ALDC enzyme in a composition comprising ALDC wherein said method comprises the step of adding a metal ion to the composition at a concentration of about 1 μM to about 200 mM, preferably about 100 μM to about 200 mM.
 50. The method of claim 49, wherein the atomic radius for the metal ion is about 140 pm to about 165 pm.
 51. The method of claim 50, wherein the atomic radius for the metal ion is about 140 pm to about 150 pm.
 52. The method of claim 51, wherein the atomic radius for the metal ion is about 142 pm to about 146 pm.
 53. The method of claim 49, wherein the metal ion is selected from the group consisting of Mg²⁺, Mn²⁺, Co²⁺, Cu²⁺, Ca²⁺, Ba²⁺, and Fe²⁺ and combinations thereof.
 54. The method of claim 53, wherein the metal ion is selected from the group consisting of Zn²⁺, Mn²⁺, and Co²⁺.
 55. The method of claim 54, wherein the metal ion is Zn²⁺.
 56. The method of claim 55, wherein the ALDC enzyme is from Lactobacillus casei, Brevibacterium acetylicum, Lactococcus lactis, Leuconostoc lactis, Enterobacter aerogenes, Bacillus subtilis, Bacillus brevis, Lactococcus lactis DX, or Bacillus licheniformis.
 57. The method of claim 55, wherein the ALDC enzyme is from Bacillus brevis or Bacillus licheniformis.
 58. The method of claim 55, wherein said ALDC enzyme has an amino acid sequence having at least 80% identity with any one selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 8 or any functional fragment thereof.
 59. A composition comprising an acetolactate decarboxylase (ALDC) enzyme and a metal ion, wherein said metal ion is present at a concentration of about 1 μM to about 200 mM, preferably about 100 μM to about 200 mM.
 60. The composition of claim 59, wherein the atomic radius for the metal ion is about 140 pm to about 165 pm.
 61. The composition of claim 59, wherein the atomic radius for the metal ion is about 140 pm to about 150 pm.
 62. The composition of claim 61, wherein the atomic radius for the metal ion is about 142 pm to about 146 pm.
 63. The composition of claim 59, wherein the metal ion is selected from the group consisting of Mg²⁺, Mn²⁺, Co²⁺, Cu²⁺, Ca²⁺, Ba²⁺, and Fe²⁺ and and combinations thereof.
 64. The composition of claim 63, wherein the metal ion is selected from the group consisting of Zn²⁺, Mn²⁺, and Co²⁺.
 65. The composition of claim 64, wherein the metal ion is Zn²⁺.
 66. A method for decomposing acetolactate comprising the step of treating a substrate with a composition comprising an ALDC enzyme and a metal ion, wherein the metal ion is present at a concentration of about 1 μM to about 200 mM in said composition; preferably said substrate is a carbohydrate containing substrate such as a wort or a fruit juice; preferably said substrate is a fermentation and/or maturation media.
 67. The method of claim 66, where the metal ion is present in said composition at a concentration of about 10 μM to about 150 mM, or about 20 μM to about 120 mM, or about 25 μM to about 100 mM, or about 25 μM to about 50 mM, or about 25 μM to about 20 mM, or about 100 μM to about 20 mM, or about 250 μM to about 20 mM, or about 1 mM to about 20 mM, or about 1 mM to about 5 mM.
 68. The method of claim 66, wherein the metal ion is present in said substrate at a concentration of: (i) about 1 μM to about 500 μM, or about 1 μM to about 300 μM, such as about 6 μM to about 300 μM, or (ii) about 1 μM to about 100 μM or about 1 μM to about 50 μM, or (iii) about 1 μM to about 25 μM, or about 6 μM to about 50 μM, or about 6 μM to about 25 μM, or about 25 μM to about 50 μM.
 69. The method of claim 66, wherein the metal ion and ALDC is added in a composition, and wherein (i) said metal ion is present in the composition at a concentration of about 1 mM to 5 mM; or (ii) the molar ratio of said metal ion to ALDC enzyme in the composition is higher than 1; or 2:1 or higher; or 10:1 or higher; or 20:1 or higher; or 30:1 or higher; or 60:1 or higher.
 70. The method of claim 69, wherein the metal ion is selected from the group consisting of Mg²⁺, Mn²⁺, Co²⁺, Cu²⁺, Ca²⁺, Ba²⁺, and Fe²⁺ and combinations thereof.
 71. The method of claim 70, wherein the metal ion is selected from the group consisting of Zn²⁺, Mn²⁺, and Co²⁺.
 72. The method of claim 71, wherein the metal ion is Zn²⁺.
 73. The method of claim 72, wherein the substrate is treated during a beer and/or wine and/or cider and/or perry and/or sake fermentation or maturation process.
 74. The method of claim 72, wherein the ALDC enzyme is from Lactobacillus casei, Brevibacterium acetylicum, Lactococcus lactis, Leuconostoc lactis, Enterobacter aerogenes, Bacillus subtilis, Bacillus brevis, Lactococcus lactis DX, or Bacillus licheniformis.
 75. The method of claim 72, wherein the ALDC enzyme is from Bacillus brevis or Bacillus licheniformis.
 76. The method of claim 72, wherein said ALDC enzyme has an amino acid sequence having at least 80% identity with any one selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 8 or any functional fragment thereof. 